Selective sulfonation of benzodiazepine derivatives

ABSTRACT

The invention relates to novel methods of preparing cell-binding agent-cytotoxic agent conjugates, wherein the cytotoxic agent is an imine-containing cytotoxic agent bearing a maleimide group. In some embodiments, the cell-binding agent (CBA) is covalently linked to the cytotoxic agent through an engineered Cys, such as an engineered Cys in the heavy chain CH3 domain, at a position corresponds to the EU/OU numbering position 442 (or C442) on an antibody CBA. The invention also provides conjugates prepared by the methods of the present invention, compositions and methods useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal using the conjugates of the invention.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.16/669,121, filed on Oct. 30, 2019, which is a continuation of U.S.patent application Ser. No. 16/364,801, filed on Mar. 26, 2019, now U.S.Pat. No. 10,494,348, which is a divisional application of U.S. patentapplication Ser. No. 15/820,648 filed on Nov. 22, 2017, now U.S. Pat.No. 10,287,256, claiming the benefit of the filing date, under 35 U.S.C.§ 119(e), of U.S. Provisional Application No. 62/425,761, filed on Nov.23, 2016. The entire content of each of these applications, includingall drawings, formulae, specification, claims and sequence listings, isincorporated herein by reference.

BACKGROUND OF THE INVENTION

Antibody-cytotoxic agent conjugates (or “antibody-drug conjugates(ADC)”) and cell binding agent-drug conjugates are emerging as apowerful class of anti-tumor agents with efficacy across a range ofcancers. Cell binding agent-drug conjugates (such as ADCs) are commonlycomposed of three distinct elements: a cell-binding agent (e.g., anantibody); a linker; and a cytotoxic moiety. The cytotoxic drug moietycan be covalently attached to lysines on the antibody, resulting inconjugates that are heterogeneous mixtures of ADCs bearing varyingnumbers of drugs attached at different positions on the antibodymolecule. Alternatively, the cytotoxic drug moiety can be covalentlylinked to cysteine thiol groups on the antibody through a thiol-reactivegroup, such as a malemide group, to form site-specific ADCs. Conjugationreactions between the antibodies and the cytotoxic agents are oftencarried out in water or an aqueous solution with small amount of anorganic solvent required for solubilizing the cytotoxic agents.

Benzodiazepine compounds, including tricyclic benzodiazepines, such aspyrrolobenzodiazepines (PBD), and tetracyclic benzodiazepines, such asindolinobenzodiazepines, have been employed as cytotoxic agents inlinkage with antibodies to generate ADCs, which have shown promisingantitumor activities. These benzodiazepine compounds contain iminebonds, which can bind to the minor groove of DNA and interfere with DNAfunction, resulting in cell death. Benzodiazepine compounds generallyhave very low solubility in water. To solubilize the benzodiazepinecompounds in the conjugation reaction with antibodies, relatively largeamount of organic solvent is required, which can de-stabilize theantibodies.

Therefore, there is a need to develop new methods for preparingconjugates of cell-binding agent and imine-containing benzodiazepinedrugs.

SUMMARY OF THE INVENTION

To improve the water solubility, the imine-containing benzodiazepinecompounds can be treated with an imine reactive reagent, such as abisulfite salt or a metabisulfite salt, before the conjugation reactionwith antibodies to form the antibody-benzodiazepine conjugates.Sulfonation of the imine group can increase the water solubility of thebenzodiazepine compounds, resulting in improved conditions for theconjugation reactions with CBAs, such as antibodies. However,nucleophilic additions between nucleophiles and α,β-unsaturatedcarbonyls are well known in synthetic organic chemistry. Thenucleophilic addition of bisulfite to the activated olefin of amaleimide is one such example of this reaction. Therefore, when theimine-containing benzodiazepine compounds bear a maleimide, the reactivegroup for covalent linkage with the antibodies, the maleimide moiety andthe imine moiety can both react with the bisulfite salt or themetabisulfite salt to form sulfonated maleimide and/or sulfonated imine.It is surprisingly found that reacting an imine-containingindolinobenzodiazepine compound bearing a maleimide group with abisulfite salt or a metabisulfite salt at a low pH can selectively andeffectively effect sulfonation of the imine group without significantsulfonation of the maleimide group, thereby increasing reaction yieldfor the conjugation reaction between the indolinobenzodiazepine compoundand the antibodies. In addition, the sulfonated indolinobenzodiazepinecompound has increased solubility in water, and as a result,significantly less amount of organic solvent (e.g., DMA) is required inthe conjugation reaction with antibodies. The presence of large amountof organic solvent in the conjugation reaction can de-stabilize theantibodies.

The present invention provides a novel method for preparing acell-binding agent-cytotoxic agent conjugate comprising animine-containing cytotoxic agent bearing a maleimide group covalentlylinked to a cell-binding agent (CBA). In some embodiments, the presentinvention provides a method of preparing a cell-binding agent-cytotoxicagent conjugate comprising the steps of:

(a) reacting an imine-moiety in an imine-containing cytotoxic agentrepresented by the following formula:

or a pharmaceutically acceptable salt thereof, with a sulfur dioxide,bisulfite salt or a metabisulfite salt in an aqueous solution at a pH of1.9 to 5.0 to form a modified cytotoxic agent comprising a modifiedimine moiety represented by the following formula:

or a pharmaceutically acceptable salt thereof; and

(b) reacting the modified cytotoxic agent with a cell-binding agent toform the cell-binding agent-cytotoxic agent conjugate,

wherein D is an imine-containing cytotoxic compound; and L is a linker.

In some embodiments, D is an imine-containing tricyclic or tetracyclicbenzodiazepine compound.

In some embodiments, D is an imine-containing tricyclic benzodiazepinecompounds.

In some embodiments, D is an imine-containing tetracyclic benzodiazepinecompounds.

As used herein, an imine-containing tricyclic benzodiazepine compoundrefers to a compound having a monocyclic ring fused to the diazepineportion of the benzodiazepine core. The monocyclic ring may contain oneor more additional heteroatoms, such as oxygen, sulfur or nitrogen, andsubstituents such as monocyclic rings or polycyclic rings. Exemplarytricyclic benzodiazepine compounds, include, but are not limited to,pyrrolobenzodiazepines (PBD), such as those described in WO2010/043880,WO2011/130616, WO2009/016516, WO 2013/177481 and WO 2012/112708.

As used herein, an imine-containing tetracyclic benzodiazepine compoundrefers to a compound having a bicyclic ring fused to the diazepineportion of the benzodiazepine core. The bicyclic ring is a fusedbicyclic ring optionally containing one or more additional heteroatoms,such as oxygen, sulfur or nitrogen. Exemplary tetracyclic benzodiazepinecompounds, include, but are not limited to, indolinobenzodiazepines(IGNs), such as those described in WO/2010/091150, and WO 2012/128868.

In some embodiments, D is an indolinobenzodiazepine.

In some embodiments, D is a pyrrolobenzodiazepine (PBD).

Also provided by the present invention is the cell-bindingagent-cytotoxic agent conjugates and modified cytotoxic agents preparedby the methods described herein.

It should be understood that any embodiments described herein, includingembodiments described only under one aspect of the invention but notother aspects, and including embodiments only appearing in the Examples,can be combined with any one or more other embodiments, unlessexplicitly disclaimed or improper.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an UPLC chromatogram of the reaction mixture ofimine-containing cytotoxic agent D1 with sodium bisulfite at pH 3.3.

FIG. 2 shows an UPLC chromatogram of the reaction mixture ofimine-containing cytotoxic agent D1 with sodium bisulfite at pH 4.75.

FIG. 3 shows an UPLC chromatogram of the reaction mixture of a PBDimine-containing cytototoxic agent talirine with sodium bisulfite.

FIGS. 4A and 4B show UPLC chromatograms of the reaction mixture of animine-containing cytotoxic agent D5 with 2.0 (FIG. 4A) or 2.5 (FIG. 4B)equivalents of sodium bisulfite.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents that can be included within the scope ofthe present invention as defined by the claims. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention.

It should be understood that any of the embodiments described herein,including those described under different aspects of the invention anddifferent parts of the specification (including embodiments describedonly in the Examples) can be combined with one or more other embodimentsof the invention, unless explicitly disclaimed or improper. Combinationof embodiments are not limited to those specific combinations claimedvia the multiple dependent claims.

Definitions

“Alkyl’ or “linear or branched alkyl” as used herein refers to asaturated linear or branched monovalent hydrocarbon radical. Inpreferred embodiments, a straight chain or branched chain alkyl hasthirty or fewer carbon atoms (e.g., C₁-C₃₀ for straight chain alkylgroup and C₃-C₃₀ for branched alkyl), and more preferably twenty orfewer carbon atoms. Even more preferably, the straight chain or branchedchain alkyl has ten or fewer carbon atoms (i.e., C₁-C₁ for straightchain alkyl group and C₃-C₁₀ for branched alkyl). In other embodiments,the straight chain or branched chain alkyl has six or fewer carbon atoms(i.e., C₁-C₆ for straight chain alky group or C₃-C₆ for branched chainalkyl). Examples of alkyl include, but are not limited to, methyl,ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, —CH₂CH(CH₃)₂),2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl 3-pentyl,2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl,1-hexyl), 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl,2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and thelike. Moreover, the term “alkyl” as used throughout the specification,examples, and claims is intended to include both “unsubstituted alkyls”and “substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. As used herein, (C_(x)-C_(xx))alkyl orC_(x-xx)alky means a linear or branched alkyl having x-xx carbon atoms.

“Alkenyl” or “linear or branched alkenyl” refers to linear orbranched-chain monovalent hydrocarbon radical of two to twenty carbonatoms with at least one site of unsaturation, i.e., a carbon-carbondouble bond, wherein the alkenyl radical includes radicals having “cis”and “trans” orientations, or alternatively, “E” and “Z” orientations.Examples include, but are not limited to, ethylenyl or vinyl (—CH═CH₂),allyl (—CH₂CH═CH₂), and the like. Preferably, the alkenyl has two to tencarbon atoms. More preferably, the alkyl has two to four carbon atoms.

“Alkynyl” or “linear or branched alkynyl” refers to a linear or branchedmonovalent hydrocarbon radical of two to twenty carbon atoms with atleast one site of unsaturation, i.e., a carbon-carbon, triple bond.Examples include, but are not limited to, ethynyl, propynyl, 1-butynyl,2-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, hexynyl, and the like.Preferably, the alkynyl has two to ten carbon atoms. More preferably,the alkynyl has two to four carbon atoms.

The terms “cyclic alkyl” and “cycloalkyl” can be used interchangeably.As used herein, the term refers to the radical of a saturatedcarbocyclic ring. In preferred embodiments, cycloalkyls have from 3 to10 carbon atoms in their ring structure, and more preferably from 5 to 7carbon atoms in the ring structure. In some embodiments, the two cyclicrings can have two or more atoms in common, e.g., the rings are “fusedrings.” Suitable cycloalkyls include, but are not limited tocycloheptyl, cyclohexyl, cyclopentyl, cyclobutyl and cyclopropyl. Insome embodiments, the cycloalkyl is a monocyclic group. In someembodiments, the cycloalkyl is a bicyclic group. In some embodiments,the cycloalkyl is a tricyclic group.

The term “cycloalklalkyl” refers to an alkyl group described above thatis substituted with a cycloalkyl group.

The term “cyclic alkenyl” refers to a carbocyclic ring radical having atleast one double bond in the ring structure.

The term “cyclic alkynyl” refers to a carbocyclic ring radical having atleast one triple bond in the ring structure.

The term “aryl” as used herein, include substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 5- to 7-membered ring, more preferably a6-membered ring. Aryl groups include, but are not limited to, phenyl,phenol, aniline, and the like. The terms “aryl” also includes“polycyclyl”, “polycycle”, and “polycyclic” ring systems having two ormore rings in which two or more atoms are common to two adjoining rings,e.g., the rings are “fused rings,” wherein at least one of the rings isaromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, or aromatic rings. In some preferredembodiments, polycycles have 2-3 rings. In certain preferredembodiments, polycyclic ring systems have two cyclic rings in which bothof the rings are aromatic. Each of the rings of the polycycle can besubstituted or unsubstituted. In certain embodiments, each ring of thepolycycle contains from 3 to 10 carbon atoms in the ring, preferablyfrom 5 to 7. For example, aryl groups include, but are not limited to,phenyl (benzene), tolyl, anthracenyl, fluorenyl, indenyl, azulenyl, andnaphthyl, as well as benzo-fused carbocyclic moieties such as5,6,7,8-tetrahydronaphthyl, and the like. In some embodiments, the arylis a single-ring aromatic group. In some embodiments, the aryl is atwo-ring aromatic group. In some embodiments, the aryl is a three-ringaromatic group.

The terms “heterocycle,” “heterocyclyl,” and “heterocyclic ring” as usedherein, refers to substituted or unsubstituted non-aromatic ringstructures of 3- to 18-membered rings, preferably 3- to 10-memberedrings, more preferably 3- to 7-membered rings, whose ring structuresinclude at least one heteroatom, preferably one to four heteroatoms,more preferably one or two heteroatoms. In certain embodiments, the ringstructure can have two cyclic rings. In some embodiments, the two cyclicrings can have two or more atoms in common, e.g., the rings are “fusedrings.” Heterocyclyl groups include, for example, piperidine,piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.Heterocycles are described in Paquette, Leo A.; “Principles of ModernHeterocyclic Chemistry” (W. A. Benjamin, New York, 1968), particularlyChapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds,A series of Monographs” (John Wiley & Sons, New York, 1950 to present),in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc.(1960) 82:5566. Examples of heterocyclic rings include, but are notlimited to, tetrahydrofurane, dihydrofuran, tetrahydrothiene,tetrahydropyran, dihydropyran, tetrahydrothiopyran, thiomorpholine,thioxane, homopiperazine, azetidine, oxetane, thietane, homopiperidine,piperidine, piperazine, pyrrolidine, morpholine, oxepane, thiepane,oxazepine, diazepine, thiazepine, 2-pyrroline, 3-pyrroline, indoline,2H-pyrane, 4H-pyrane, dioxane, 1,3-dioxolane, pyrazoline, dithiane,dithiolane, dihydropyrane, dihydrothiene, dihydrofurane,pyrazolidinylimidazoline, imidazolidine, 3-azabicyco[3.1.0]hexane,3-azabicyclo[4.1.0]heptane, and azabicyclo[2.2.2]hexane. Spiro moietiesare also included within the scope of this definition. Examples of aheterocyclic group wherein ring atoms are substituted with oxo (═O)moieties are pyrimidinone and 1,1-dioxo-thiomorpholine.

The term “heteroaryl” as used herein, refers to substituted orunsubstituted aromatic single ring structures, preferably 5- to7-membered rings, more preferably 5- to 6-membered rings, whose ringstructures include at least one heteroatom (e.g., 0, N, or S),preferably one to four or one to three heteroatoms, more preferably oneor two heteroatoms. When two or more heteroatoms are present in aheteroaryl ring, they may be the same or different. The term“heteroaryl” also includes “polycyclyl”, “polycycle”, and “polycyclic”ring systems having two or more cyclic rings in which two or more ringatoms are common to two adjoining rings, e.g., the rings are “fusedrings,” wherein at least one of the rings is heteroaromatic, e.g., theother cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls, heteroaromatics, and/or heterocyclyls. In some preferredembodiments, polycyclic heteroaryls have 2-3 rings. In certainembodiments, preferred polycyclic heteroaryls have two cyclic rings inwhich both of the rings are aromatic. In certain embodiments, each ringof the polycycle contains from 3 to 10 atoms in the ring, preferablyfrom 5 to 7 atoms in the ring. For examples, heteroaryl groups include,but are not limited to, pyrrole, furan, thiophene, imidazole, oxazole,thiazole, pyrazole, pyridine, pyrazine, pyridazine, quinoline,pyrimidine, indolizine, indole, indazole, benzimidazole, benzothiazole,benzofuran, benzothiophene, cinnoline, phthalazine, quinazoline,carbazole, phenoxazine, quinoline, purine and the like. In someembodiments, the heteroaryl is a single-ring aromatic group. In someembodiments, the heteroaryl is a two-ring aromatic group. In someembodiments, the heteroaryl is a three-ring aromatic group.

The heterocycle or heteroaryl groups can be carbon (carbon-linked) ornitrogen (nitrogen-linked) attached where such is possible. By way ofexample and not limitation, carbon bonded heterocycles or heteroarylsare bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5,or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or O-carboline.

The heteroatoms present in heteroaryl or heterocyclyl include theoxidized forms such as NO, SO, and SO₂.

The term “halo” or “halogen” refers to fluorine (F), chlorine (C₁),bromine (Br) or iodine (I).

As used herein, the term “haloalkyl” refers to an alkyl, as definedherein, that is substituted by one or more halo groups as definedherein. The haloalkyl can be monohaloalkyl, dihaloalkyl orpolyhaloalkyl. A monohaloalkyl can have one fluoro, chloro, bromo, oriodo substituent. Dihaloalkyl or polyhaloalkyl can be substituted withtwo or more of the same halo atoms or a combination of different halogroups. Examples of haloalkyl include, but are not limited to,flouromethyl, difluoromethyl, trifluoromethyl, chloromethyl,dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl,diflurochloromethyl, dichlorofluoromethyl, difluoroethyl,diflouropropyl, dichloroethyl and dichloropropyl.

“Alkoxy” used herein refers to alkyl-O—, wherein alkyl is defined hereinabove. Examples of alkoxy include, not are not limited to, methoxy,ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy,and the like.

The alkyl, haloalkyl, alkoxy, alkenyl, alkynyl, cyclic alkyl, cyclicalkenyl, cyclic alkynyl, carbocyclyl, aryl, heterocyclyl and heteroaryldescribed above can be optionally substituted with one or more (e.g., 2,3, 4, 5, 6 or more) substituents.

Unless specifically stated as “unsubstituted,” references to chemicalmoieties herein are understood to also include substituted variants. Forexample, reference to an “alkyl” group or moiety implicitly includesboth substituted and unsubstituted variants. Examples of substituents onchemical moieties includes but is not limited to, halogen, hydroxyl,carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl),thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl,alkylthio, acyloxy, phosphoryl, phosphate, phosphonate, amino, amido,amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate,sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, oraryl or heteroaryl moiety.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the application includesinstances where the circumstance occurs and instances where it does not.For example, the phrase “optionally substituted” means that anonhydrogen substituent may or may not be present on a given atom, and,thus, the application includes structures wherein a non-hydrogensubstituent is present and structures wherein a nonhydrogen substituentis not present.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons, nitrogens, oxygens or sulfurs atoms.It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc. As used herein, the term “substituted” iscontemplated to include all permissible substituents of organiccompounds. In a broad aspect, the permissible substituents includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and non-aromatic substituents of organiccompounds. The permissible substituents can be one or more and the sameor different for appropriate organic compounds. For purposes of theinvention, the heteroatoms such as nitrogen may have hydrogensubstituents and/or any permissible substituents of organic compoundsdescribed herein which satisfy the valences of the heteroatoms.Substituents can include any substituents described herein, for example,a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, an alkylthio,an acyloxy, a phosphoryl, a phosphate, a phosphonate, an amino, anamido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl,an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromaticmoiety. To illustrate, monofluoroalkyl is alkyl substituted with afluoro substituent, and difluoroalkyl is alkyl substituted with twofluoro substituents. It should be recognized that if there is more thanone substitution on a substituent, each non-hydrogen substituent may beidentical or different (unless otherwise stated).

If a carbon of a substituent is described as being optionallysubstituted with one or more of a list of substituents, one or more ofthe hydrogens on the carbon (to the extent there are any) can separatelyand/or together be replaced with an independently selected optionalsubstituent. If a nitrogen of a substituent is described as beingoptionally substituted with one or more of a list of substituents, oneor more of the hydrogens on the nitrogen (to the extent there are any)can each be replaced with an independently selected optionalsubstituent. One exemplary substituent can be depicted as —NR′R″,wherein R′ and R″ together with the nitrogen atom to which they areattached, can form a heterocyclic ring. The heterocyclic ring formedfrom R′ and R″ together with the nitrogen atom to which they areattached can be partially or fully saturated. In some embodiments, theheterocyclic ring consists of 3 to 7 atoms. In other embodiments, theheterocyclic ring is selected from the group consisting of pyrrolyl,imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, pyridyl andthiazolyl.

This specification uses the terms “substituent,” “radical,” and “group”interchangeably.

If a group of substituents are collectively described as beingoptionally substituted by one or more of a list of substituents, thegroup can include: (1) unsubstitutable substituents, (2) substitutablesubstituents that are not substituted by the optional substituents,and/or (3) substitutable substituents that are substituted by one ormore of the optional substituents.

If a substituent is described as being optionally substituted with up toa particular number of non-hydrogen substituents, that substituent canbe either (1) not substituted; or (2) substituted by up to thatparticular number of non-hydrogen substituents or by up to the maximumnumber of substitutable positions on the substituent, whichever is less.Thus, for example, if a substituent is described as a heteroaryloptionally substituted with up to 3 non-hydrogen substituents, then anyheteroaryl with less than 3 substitutable positions would be optionallysubstituted by up to only as many non-hydrogen substituents as theheteroaryl has substitutable positions. Such substituents, innon-limiting examples, can be selected from a linear, branched or cyclicalkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, aryl,heteroaryl, heterocyclylyl, halogen, guanidinium [—NH(C═NH)NH₂], —OR¹⁰⁰,NR¹⁰¹R¹⁰², —NO₂, —NR¹⁰¹COR¹⁰², —SR¹⁰⁰, a sulfoxide represented by—SOR¹⁰¹, a sulfone represented by —SO₂R¹⁰¹, a sulfonate —SO₃M, a sulfate—OSO₃M, a sulfonamide represented by —SO₂NR¹⁰¹R¹⁰², cyano, an azido,—COR¹⁰¹, —OCOR¹⁰¹, —OCONR¹⁰¹R¹⁰² and a polyethylene glycol unit(—OCH₂CH₂)_(n)R¹⁰¹ wherein M is H or a cation (such as Na⁺ or K⁺); R¹⁰¹,R¹⁰² and R¹⁰³ are each independently selected from H, linear, branchedor cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, apolyethylene glycol unit (—OCH₂CH₂)n-R¹⁰⁴, wherein n is an integer from1 to 24, an aryl having from 6 to 10 carbon atoms, a heterocyclic ringhaving from 3 to 10 carbon atoms and a heteroaryl having 5 to 10 carbonatoms; and R¹⁰⁴ is H or a linear or branched alkyl having 1 to 4 carbonatoms, wherein the alkyl, alkenyl, alkynyl, aryl, heteroaryl andheterocyclcyl in the groups represented by R¹⁰⁰, R¹⁰¹, R¹⁰², R¹⁰³ andR¹⁰⁴ are optionally substituted with one or more (e.g., 2, 3, 4, 5, 6 ormore) substituents independently selected from halogen, —OH, —CN, —NO₂and unsubstituted linear or branched alkyl having 1 to 4 carbon atoms.Preferably, the substituents for the optionally substituted alkyl,alkenyl, alkynyl, cyclic alkyl, cyclic alkenyl, cyclic alkynyl,carbocyclyl, aryl, heterocyclyl and heteroaryl described above includehalogen, —CN, —NR¹⁰²R¹⁰³, —CF₃, —OR¹⁰¹, aryl, heteroaryl, heterocyclyl,—SR¹⁰¹, —SOR¹⁰¹, —SO₂R¹⁰¹ and —SO₃M.

The number of carbon atoms in a group can be specified herein by theprefix “C_(x-xx)” or “C_(x)-C_(xx)”, wherein x and xx are integers. Forexample, “C₁₋₄alkyl” or “C₁-C₄ alkyl” is an alkyl group having from 1 to4 carbon atoms.

The term “compound” or “cytotoxic compound,” “cytotoxic dimer” and“cytotoxic dimer compound” are used interchangeably. They are intendedto include compounds for which a structure or formula or any derivativethereof has been disclosed in the present invention or a structure orformula or any derivative thereof that has been incorporated byreference. The term also includes, stereoisomers, geometric isomers,tautomers, solvates, metabolites, salts (e.g., pharmaceuticallyacceptable salts) and prodrugs, and prodrug salts of a compound of allthe formulae disclosed in the present invention. The term also includesany solvates, hydrates, and polymorphs of any of the foregoing. Thespecific recitation of “stereoisomers,” “geometric isomers,”“tautomers,” “solvates,” “metabolites,” “salt” “prodrug,” “prodrugsalt,” “conjugates,” “conjugates salt,” “solvate,” “hydrate,” or“polymorph” in certain aspects of the invention described in thisapplication shall not be interpreted as an intended omission of theseforms in other aspects of the invention where the term “compound” isused without recitation of these other forms.

The term “conjugate” as used herein refers to a compound describedherein or a derivative thereof that is linked to a cell binding agent.

The term “linkable to a cell binding agent” as used herein refers to thecompounds described herein or derivatives thereof comprising at leastone linking group or a precursor thereof suitable to bond thesecompounds or derivatives thereof to a cell binding agent.

The term “precursor” of a given group refers to any group that can leadto that group by any deprotection, a chemical modification, or acoupling reaction.

The term “chiral” refers to molecules that have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules that are superimposable on their mirrorimage partner.

The term “stereoisomer” refers to compounds that have identical chemicalconstitution and connectivity, but different orientations of their atomsin space that cannot be interconverted by rotation about single bonds.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers can separate under high resolution analytical proceduressuch as crystallization, electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound that arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds,” John Wiley & Sons, Inc., NewYork, 1994. The compounds of the invention can contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in opticallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand I or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or 1 meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer can also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which canoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies that are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

As used herein, a “benzodiazepine” compound is a compound having abenzodiazepine core structure. The benzodiazepine core can besubstituted or unsubstituted, and/or fused with one or more ringstructures. It also includes a compound having two benzodiazepine corelinked by a linker. The imine functionality (—C═N—) as part ofbenzodiazepine core can be reduced.

As used herein, a “pyrrolobenzodiazepine” (PBD) compound is a compoundhaving a pyrrolobenzodiazepine core structure. The pyrrolobenzodiazepinecan be substituted or unsubstituted. It also includes a compound havingtwo pyrrolobenzodiazepine core linked by a linker. The iminefunctionality (—C═N—) as part of indolinobenzodiazepine core can bereduced.

In certain embodiments, the pyrrolobenzodiazepine compound comprises acore structure represented by

which can be optionally substituted.

In certain embodiments, the pyrrolobenzodiazepine compounds comprises acore structure represented by

which can be optionally substituted.

As used herein, a “indolinobenzodiazepine” (IGN) compound is a compoundhaving an indolinobenzodiazepine core structure. Theindolinobenzodiazepine can be substituted or unsubstituted. It alsoincludes a compound having two indolinobenzodiazepine core linked by alinker. The imine functionality (—C═N—) as part ofindolinobenzodiazepine core can be reduced.

In certain embodiments, the indolinobenzodiazepine compound comprises acore structure represented by

which can be optionally substituted.

In some embodiments, the indolinobenzodiazepine compound comprises acore structure represented by

which can be further substituted.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe invention. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate “mesylate,” ethanesulfonate, benzenesulfonate,p-toluenesulfonate, pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g.,sodium and potassium) salts, alkaline earth metal (e.g., magnesium)salts, and ammonium salts. A pharmaceutically acceptable salt caninvolve the inclusion of another molecule such as an acetate ion, asuccinate ion or other counter ion. The counter ion can be any organicor inorganic moiety that stabilizes the charge on the parent compound.Furthermore, a pharmaceutically acceptable salt can have more than onecharged atom in its structure. Instances where multiple charged atomsare part of the pharmaceutically acceptable salt can have multiplecounter ions. Hence, a pharmaceutically acceptable salt can have one ormore charged atoms and/or one or more counter ion.

If the compound of the invention is a base, the desired pharmaceuticallyacceptable salt can be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,methanesulfonic acid, phosphoric acid and the like, or with an organicacid, such as acetic acid, maleic acid, succinic acid, mandelic acid,fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid,salicylic acid, a pyranosidyl acid, such as glucuronic acid orgalacturonic acid, an alpha hydroxy acid, such as citric acid ortartaric acid, an amino acid, such as aspartic acid or glutamic acid, anaromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid,such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.

If the compound of the invention is an acid, the desiredpharmaceutically acceptable salt can be prepared by any suitable method,for example, treatment of the free acid with an inorganic or organicbase, such as an amine (primary, secondary or tertiary), an alkali metalhydroxide or alkaline earth metal hydroxide, or the like. Illustrativeexamples of suitable salts include, but are not limited to, organicsalts derived from amino acids, such as glycine and arginine, ammonia,primary, secondary, and tertiary amines, and cyclic amines, such aspiperidine, morpholine and piperazine, and inorganic salts derived fromsodium, calcium, potassium, magnesium, manganese, iron, copper, zinc,aluminum and lithium.

As used herein, the term “solvate” means a compound that furtherincludes a stoichiometric or non-stoichiometric amount of solvent suchas water, isopropanol, acetone, ethanol, methanol, DMSO, ethyl acetate,acetic acid, and ethanolamine dichloromethane, 2-propanol, or the like,bound by non-covalent intermolecular forces. Solvates or hydrates of thecompounds are readily prepared by addition of at least one molarequivalent of a hydroxylic solvent such as methanol, ethanol,1-propanol, 2-propanol or water to the compound to result in solvationor hydration of the imine moiety.

The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the mammal beingtreated therewith.

The term “protecting group” or “protecting moiety” refers to asubstituent that is commonly employed to block or protect a particularfunctionality while reacting other functional groups on the compound, aderivative thereof, or a conjugate thereof. For example, an“amine-protecting group” or an “amino-protecting moiety” is asubstituent attached to an amino group that blocks or protects the aminofunctionality in the compound. Such groups are well known in the art(see for example P. Wuts and T. Greene, 2007, Protective Groups inOrganic Synthesis, Chapter 7, J. Wiley & Sons, NJ) and exemplified bycarbamates such as methyl and ethyl carbamate, FMOC, substituted ethylcarbamates, carbamates cleaved by 1,6-β-elimination (also termed “selfimmolative”), ureas, amides, peptides, alkyl and aryl derivatives.Suitable amino-protecting groups include acetyl, trifluoroacetyl,t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and9-fluorenylmethylenoxycarbonyl (Fmoc). For a general description ofprotecting groups and their use, see P. G. M. Wuts & T. W. Greene,Protective Groups in Organic Synthesis, John Wiley & Sons, New York,2007.

The term “leaving group” refers to an group of charged or unchargedmoiety that departs during a substitution or displacement. Such leavinggroups are well known in the art and include, but not limited to,halogens, esters, alkoxy, hydroxyl, tosylates, triflates, mesylates,nitriles, azide, carbamate, disulfides, thioesters, thioethers anddiazonium compounds.

The term “bifunctional crosslinking agent,” “bifunctional linker” or“crosslinking agents” refers to modifying agents that possess tworeactive groups; one of which is capable of reacting with a cell bindingagent while the other one reacts with the cytotoxic compound to link thetwo moieties together. Such bifunctional crosslinkers are well known inthe art (see, for example, Isalm and Dent in Bioconjugation chapter 5, p218-363, Groves Dictionaries Inc. New York, 1999). For example,bifunctional crosslinking agents that enable linkage via a thioetherbond includeN-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) tointroduce maleimido groups, or withN-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB) to introduceiodoacetyl groups. Other bifunctional crosslinking agents that introducemaleimido groups or haloacetyl groups on to a cell binding agent arewell known in the art (see US Patent Applications 2008/0050310,20050169933, available from Pierce Biotechnology Inc. P.O. Box 117,Rockland, Ill. 61105, USA) and include, but not limited to,bis-maleimidopolyethyleneglycol (BMPEO), BM(PEO)₂, BM(PEO)₃,N-(β-maleimidopropyloxy)succinimide ester (BMPS), γ-maleimidobutyricacid N-succinimidyl ester (GMBS), ε-maleimidocaproic acidN-hydroxysuccinimide ester (EMCS), 5-maleimidovaleric acid NHS, HBVS,N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate),which is a “long chain” analog of SMCC (LC-SMCC),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),4-(4-N-maleimidophenyl)-butyric acid hydrazide or HCl salt (MPBH),N-succinimidyl 3-(bromoacetamido)propionate (SBAP), N-succinimidyliodoacetate (SIA), κ-maleimidoundecanoic acid N-succinimidyl ester(KMUA), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB),succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH),succinimidyl-(4-vinylsulfonyl)benzoate (SVSB), dithiobis-maleimidoethane(DTME), 1,4-bis-maleimidobutane (BMB), 1,4bismaleimidyl-2,3-dihydroxybutane (BMDB), bis-maleimidohexane (BMH),bis-maleimidoethane (BMOE), sulfosuccinimidyl4-(N-maleimido-methyl)cyclohexane-1-carboxylate (sulfo-SMCC),sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate (sulfo-SIAB),m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS),N-(γ-maleimidobutryloxy)sulfosuccinimde ester (sulfo-GMBS),N-(ε-maleimidocaproyloxy)sulfosuccimido ester (sulfo-EMCS),N-(κ-maleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS), andsulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB).

Heterobifunctional crosslinking agents are bifunctional crosslinkingagents having two different reactive groups. Heterobifunctionalcrosslinking agents containing both an amine-reactiveN-hydroxysuccinimide group (NHS group) and a carbonyl-reactive hydrazinegroup can also be used to link the cytotoxic compounds described hereinwith a cell-binding agent (e.g., antibody). Examples of suchcommercially available heterobifunctional crosslinking agents includesuccinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH),succinimidyl 4-hydrazidoterephthalate hydrochloride (SHTH) andsuccinimidyl hydrazinium nicotinate hydrochloride (SHNH). Conjugatesbearing an acid-labile linkage can also be prepared using ahydrazine-bearing benzodiazepine derivative of the present invention.Examples of bifunctional crosslinking agents that can be used includesuccinimidyl-p-formyl benzoate (SFB) andsuccinimidyl-p-formylphenoxyacetate (SFPA).

Bifunctional crosslinking agents that enable the linkage of cell bindingagent with cytotoxic compounds via disulfide bonds are known in the artand include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP),N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB),N-succinimidyl-4-(2-pyridyldithio)2-sulfo butanoate (sulfo-SPDB) tointroduce dithiopyridyl groups. Other bifunctional crosslinking agentsthat can be used to introduce disulfide groups are known in the art andare disclosed in U.S. Pat. Nos. 6,913,748, 6,716,821 and US PatentPublications 20090274713 and 20100129314, all of which are incorporatedherein by reference. Alternatively, crosslinking agents such as2-iminothiolane, homocysteine thiolactone or S-acetylsuccinic anhydridethat introduce thiol groups can also be used.

A “linker,” “linker moiety,” or “linking group” as defined herein refersto a moiety that connects two groups, such as a cell binding agent and acytotoxic compound, together. Typically, the linker is substantiallyinert under conditions for which the two groups it is connecting arelinked. A bifunctional crosslinking agent can comprise two reactivegroups, one at each ends of a linker moiety, such that one reactivegroup can be first reacted with the cytotoxic compound to provide acompound bearing the linker moiety and a second reactive group, whichcan then react with a cell binding agent. Alternatively, one end of thebifunctional crosslinking agent can be first reacted with the cellbinding agent to provide a cell binding agent bearing a linker moietyand a second reactive group, which can then react with a cytotoxiccompound. The linking moiety can contain a chemical bond that allows forthe release of the cytotoxic moiety at a particular site. Suitablechemical bonds are well known in the art and include disulfide bonds,thioether bonds, acid labile bonds, photolabile bonds, peptidase labilebonds and esterase labile bonds (see for example U.S. Pat. Nos.5,208,020; 5,475,092; 6,441,163; 6,716,821; 6,913,748; 7,276,497;7,276,499; 7,368,565; 7,388,026 and 7,414,073). Preferred are disulfidebonds, thioether and peptidase labile bonds. Other linkers that can beused in the present invention include non-cleavable linkers, such asthose described in are described in detail in U.S. publication number20050169933, or charged linkers or hydrophilic linkers and are describedin US 2009/0274713, US 2010/01293140 and WO 2009/134976, each of whichis expressly incorporated herein by reference, each of which isexpressly incorporated herein by reference.

The term “amino acid” refers to naturally occurring amino acids ornon-naturally occurring amino acid. In some embodiments, the amino acidis represented by NH₂—C(R^(aa′)R^(aa))—C(═O)OH, wherein R^(aa) andR^(aa′) are each independently H, an optionally substituted linear,branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbonatoms, aryl, heteroaryl or heterocyclyl or R^(aa) and the N-terminalnitrogen atom can together form a heterocyclic ring (e.g., as inproline). The term “amino acid residue” refers to the correspondingresidue when one hydrogen atom is removed from the amine and/or carboxyend of the amino acid, such as —NH—C(R^(aa′)R^(aa))—C(═O)O—.

The term “peptide” refers to short chains of amino acid monomers linkedby peptide (amide) bonds. In some embodiments, the peptides contain 2 to20 amino acid residues. In other embodiments, the peptides contain 2 to10 amino acid residues. In yet other embodiments, the peptides contain 2to 5 amino acid residues. As used herein, when a peptide is a portion ofa cytotoxic agent or a linker described herein represented by a specificsequence of amino acids, the peptide can be connected to the rest of thecytotoxic agent or the linker in both directions. For example, adipeptide X₁-X₂ includes X₁-X₂ and X₂—X₁. Similarly, a tripeptideX₁-X₂-X₃ includes X₁-X₂-X₃ and X₃—X₂—X₁ and a tetrapeptide X₁-X₂-X₃-X₄includes X₁-X₂-X₃-X₄ and X₄—X₂—X₃—X₁. X₁, X₂, X₃ and X₄ represents anamino acid residue.

The term “cation” refers to an ion with positive charge. The cation canbe monovalent (e.g., Na⁺, K⁺, etc.), bi-valent (e.g., Ca²⁺, Mg²⁺, etc.)or multi-valent (e.g., Al³⁺ etc.). Preferably, the cation is monovalent.

The term “cysteine engineered antibody” includes an antibody with atleast one cysteine (Cys) that is not normally present at a given residueof the antibody light chain or heavy chain. Such Cys, which may also bereferred to as “engineered Cys,” can be introduced, for example, bystandard recombinant technology (e.g., by replacing the coding sequencefor a non-Cys residue at the target residue with a coding sequence forCys). In certain embodiments, the Cys engineered antibody of theinvention has an engineered Cys in the heavy chain. In certainembodiments, the engineered Cys is in or near the CH3 domain of theheavy chain. In certain embodiments, the engineered Cys is at residue442 of the heavy chain (EU/OU numbering).

As used herein, all antibody amino acid residues described herein arenumbered according to the EU index, Kabat et al., Sequences of Proteinsof Immunological Interest, 5^(th) Ed., NIH publication No. 91-3242, 1991(EU/OU numbering, entire content incorporated herein by reference). Thecommon isotypes are referred to as G1, G2, G4, etc.

The C442 residue can be conjugated with a cytotoxic drug/agent throughthe free thiol group of the C442 residue, such as through reacting witha thiol-reactive agent of the cytotoxic drug (e.g., a maleimido group).

As used herein, an “aqueous solution” refers to a solution in which thesolvent is water or a mixture of water and one or more organic solvents.

As used herein, the term “treating” or “treatment” includes reversing,reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a condition in manner to improve or stabilize a subject'scondition. As used herein, and as well understood in the art “treatment”is an approach for obtaining beneficial or desired results, includingclinical results. Beneficial or desired clinical results can include,but are not limited to, alleviation, amelioration, or slowing theprogression, of one or more symptoms or conditions associated with acondition, e.g., cancer, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Exemplary beneficialclinical results are described herein.

Methods of the Present Invention

The present invention provides novel methods for preparing acell-binding agent-cytotoxic agent conjugate comprising animine-containing cytotoxic agent bearing a maleimide group covalentlylinked to a cell-binding agent (CBA).

In some embodiments, the methods of the present invention for preparinga cell-binding agent-cytotoxic agent conjugate comprise the steps of:

(a) reacting an imine-moiety in an imine-containing cytotoxic agentrepresented by the following formula:

or a pharmaceutically acceptable salt thereof, with sulfur dioxide, abisulfite salt or a metabisulfite salt in an aqueous solution at a pH of1.9 to 5.0 to form a modified cytotoxic agent comprising a modifiedimine moiety represented by the following formula:

or a pharmaceutically acceptable salt thereof; and

(b) reacting the modified cytotoxic agent with a cell-binding agent toform the cell-binding agent-cytotoxic agent conjugate. In someembodiments, the bisulfite salt is sodium bisulfite or potassiumbisulfite. More specifically, the bisulfite salt is sodium bisulfite. Inyet other embodiments, the metabisulfite salt is sodium metabisulfite orpotassium metabisulfite. More specifically, the metabisulfite salt issodium metabisulfite.

The present invention also provides a method of preparing a modifiedcytotoxic agent comprising the step of reacting an imine-moiety in animine-containing cytotoxic agent represented by the following formula:

or a pharmaceutically acceptable salt thereof, with sulfur dioxide, abisulfite salt or a metabisulfite salt in an aqueous solution at a pH of1.9 to 5.0 to form a modified cytotoxic agent comprising a modifiedimine moiety represented by the following formula:

or a pharmaceutically acceptable salt thereof. In some embodiments, thereaction can be carried out under the reaction conditions described inthe 1^(st), 2^(nd), 3^(rd), 4^(th), 5^(th), 6^(th) or 7^(th) aspectbelow.

In a 1^(st) aspect, for the method of the present invention describedabove, the reaction of step (a) is carried out at a pH of 1.9 to 5.0.More specifically, the pH is 2.5 to 4.9, 1.9 to 4.8, 2.0 to 4.8, 2.5 to4.5, 2.9 to 4.5, 2.9 to 4.0, 2.9 to 3.7, 3.1 to 3.5, or 3.2 to 3.4. Inanother specific embodiment, the reaction of step (a) is carried out ata pH of 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.5, 4.6,4.7, 4.8, 4.9 or 5.0. In yet another specific embodiment, the reactionof step (a) is carried out at a pH of 3.3.

As used herein, a specific pH value means the specific value 0.05.

In some embodiments, the reaction of step (a) is carried out in thepresence of a buffer solution. Any suitable buffer solution known in theart can be used in the methods of the present invention. Suitable buffersolutions include, for example, but are not limited to, a citratebuffer, an acetate buffer, a succinate buffer, a phosphate buffer, aglycine-containing buffer (e.g., glycine-HCl buffer), a phthalate buffer(e.g., a buffer solution comprising sodium or potassium hydrogenphthalate), and a combination thereof. In some embodiments, the buffersolution is a succinate buffer. In some embodiments, the buffer solutionis a phosphate buffer. In some embodiments, the buffer is acitrate-phosphate buffer. In some embodiments, the buffer is acitrate-phosphate buffer comprising citric acid and Na₂HPO₄. In otherembodiments, the buffer is a citrate-phosphate buffer comprising citricacid and K₂HPO₄. In some embodiments, the concentration of the buffersolution described above can be in the range of 10 to 250 mM, 10 to 200mM, 10 to 150 mM, 10 to 100 mM, 25 to 100 mM, 25 to 75 mM, 10 to 50 mM,or 20 to 50 mM.

In a 2^(nd) aspect, the reaction step (a) is carried out in the absenceof a buffer solution (e.g., the buffers described in the 1^(st) aspect).In some embodiments, the present method comprises the steps of (a)reacting an imine-moiety in an imine-containing cytotoxic agentrepresented by formula (A) or a pharmaceutically acceptable saltthereof, with sulfur dioxide, a bisulfite salt or a metabisulfite saltin an aqueous solution to form a modified cytotoxic agent comprising amodified imine moiety represented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein the aqueoussolution does not comprise a buffer; and

(b) reacting the modified cytotoxic agent with a cell-binding agent toform the cell-binding agent-cytotoxic agent conjugate. In someembodiments, the reaction of step (a) is carried out in a mixture of anorganic solvent and water. More specifically, the reaction of step (a)is carried out in a mixture of dimethyacetamide (DMA) and water. In someembodiments, the mixture of DMA and water comprises less than 60% of DMAby volume. Even more specifically, the volume ratio of DMA and water is1:1.

In a 3^(rd) aspect, for the methods described above or in the 1^(st) or2^(nd) aspect, 0.5 to 5.0 equivalents of the bisulfite salt or 0.25 or2.5 equivalents of the metabisulfite salt is used for every 1 equivalentof the imine-containing cytotoxic agent in the reaction of step (a). Insome embodiments, 0.5 to 4.5, 0.5 to 4.0, 0.5 to 3.5, 0.5 to 4.0, 0.5 to3.5, 0.5 to 3.0, 0.5 to 2.5, 0.8 to 2.0, 0.9 to 1.8, 1.0 to 1.7, 1.1 to1.6, or 1.2 to 1.5 equivalents of the bisulfite salt or 0.25 to 2.25,0.25 to 2.0, 0.25 to 1.75, 0.25 to 2.0, 0.25 to 1.75, 0.25 to 1.5, 0.25to 1.25, 0.4 to 1.0, 0.45 to 0.9, 0.5 to 0.85, 0.55 to 0.8, or 0.6 to0.75 equivalents of the metabisulfite salt is used for every 1equivalent of the imine-containing cytotoxic agent. In otherembodiments, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0,3.1, 3.2, 3.3, 3.4, 3.5, 4.0, 4.5 or 5.0 equivalents of the bisulfitesalt or 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65 0.7, 0.75, 0.8,0.85, 0.9, 0.95, 1.0, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45,1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 2.0, 2.25 or 2.5 equivalents of themetabisulfite salt is used for every 1 equivalent of theimine-containing cytotoxic agent. In yet other embodiments, 1.4equivalents of the bisulfite salt or 0.7 equivalent of the metabisulfitesalt is used for every 1 equivalent of the imine-containing cytotoxicagent. In other embodiments, 1.2 equivalents of the bisulfite salt or0.6 equivalent of the metabisulfite salt is used for every 1 equivalentof the imine-containing cytotoxic agent.

As used herein, a specific equivalent means the specific value 0.05.

In a 4^(th) aspect, for methods of the present invention, the reactionof step (a) is carried out at a pH of 2.9 to 3.7 and 1.0 to 1.8equivalents of the bisulfite salt or 0.5 to 0.9 equivalents of themetabisulfite salt is reacted with 1 equivalent of the imine-containingcytotoxic agent. In some embodiments, the reaction of step (a) iscarried out at a pH of 3.1 to 3.5 and 1.1 to 1.6 equivalents of thebisulfite salt or 0.55 to 0.8 equivalents of the metabisulfite salt isreacted with 1 equivalent of the imine-containing cytotoxic agent. Inother embodiments, the reaction of step (a) is carried out at a pH of3.2 to 3.4 and 1.3 to 1.5 equivalents of the bisulfite salt or 0.65 to0.75 equivalents of the metabisulfite is reacted with 1 equivalent ofthe imine-containing cytotoxic agent. In other embodiments, the reactionof step (a) is carried out at a pH of 3.3 and 1.4 equivalents of thebisulfite salt or 0.7 equivalent of the metabisulfite salt is reactedwith 1 equivalent of the imine-containing cytotoxic agent. In yet otherembodiments, In other embodiments, the reaction of step (a) is carriedout at a pH of 3.3 and 1.4 equivalents of sodium bisulfite is reactedwith 1 equivalent of the imine-containing cytotoxic agent.

In a 5^(th) aspect, for the methods of the present invention describedherein or in the 1^(st), 2^(nd), 3^(rd) or 4^(th) aspect, the reactionof step (a) is carried out in a mixture of an organic solvent and water.Any suitable organic solvent can be used. Exemplary organic solventsinclude, but are not limited to, alcohols (e.g., methanol, ethanol,propanol, etc.), dimethylformamide (DMF), dimethylsulfoxide (DMSO),acetonitrile, acetone, methylene chloride, etc. In some embodiments, theorganic solvent is miscible with water. In other embodiments, theorganic solvent is not miscible with water, i.e., the reaction of step(a) is carried out in a biphasic solution. In some embodiments, theorganic solvent is dimethylacetamide (DMA). The organic solvent (e.g.,DMA) can be present in the amount of 1%-99%, 1-95%, 10-80%, 20-70%,30-70%, 1-60%, 5-60%, 10-60%, 20-60%, 30-60%, 40-60%, 45-55%, 10-50%, or20-40%, by volume of the total volume of water and the organic solvent.In some embodiments, the reaction of step (a) is carried out in amixture of DMA and water, wherein the volume ratio of DMA and water is1:1.

In a 6^(th) aspect, for the methods of the present invention describedherein or in the 1^(st), 2^(nd), 3^(rd), 4^(th) or 5^(th) aspect, thereaction of step (a) can be carried out at any suitable temperature. Insome embodiments, the reaction is carried out at a temperature from 0°C. to 50° C., from 10° C. to 50° C., from 10° C. to 40° C., or from 10°C. to 30° C. In other embodiments, the reaction is carried out at atemperature from 15° C. to 30° C., from 20° C. to 30° C., from 15° C. to25° C., from 16° C. to 24° C., from 17° C. to 23° C., from 18° C. to 22°C. or from 19° C. to 21° C. In yet other embodiments, the reaction canbe carried out at 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21°C., 22° C., 23° C., 24° C. or 25° C. In some embodiments, the reactioncan be carried out from 0° C. to 15° C., from 0° C. to 10° C., from 1°C. to 10° C., 5° C. to 15° C., or from 5° C. to 10° C.

In a 7^(th) aspect, for the methods of the present invention describedherein or in the 1^(st), 2^(nd), 3^(rd), 4^(th), 5^(th) or 6^(th)aspect, the reaction of step (a) is carried out for 1 minute to 48hours, 5 minutes to 36 hours, 10 minutes to 24 hours, 30 minutes to 24hours, 30 minutes to 20 hours, 1 hour to 20 hours, 1 hour to 15 hours, 1hour to 10 hours, 2 hours to 10 hours, 3 hours to 9 hours, 3 hours to 8hours, 4 hours to 6 hours, or 1 hour to 4 hours. In some embodiments,the reaction is allowed to proceed for 4 to 6 hours. In otherembodiments, the reaction is allowed to proceed for 10 minutes, 15minutes, 20 minutes, 30 minutes, 1 hours, 2 hours, 3 hours, 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours,13 hours, 14 hours, 15 hours, etc. In other embodiments, the reaction isallowed to proceed for 4 hours. In yet other embodiments, the reactionis allowed to proceed for 2 hours.

In a 8^(th) aspect, for the methods of the present invention describedherein or in the 1^(st), 2^(nd), 3^(rd), 4^(th), 5^(th), 6^(th) or7^(th) aspect, the reaction of step (b) is carried out at a pH of 4 to9. In some embodiments, the reaction of step (b) is carried out at a pHof 4.5 to 8.5, 5 to 8.5, 5 to 8, 5 to 7.5, 5 to 7, 5 to 6.5, or 5.5 to6.5. In other embodiments, the reaction of step (b) is carried out at pH5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3,6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, or 8.0.

In some embodiments, for the methods of the present invention describedherein or in the 1^(st), 2^(nd), 3^(rd), 4^(th), 5^(th), 6^(th), 7^(th)or 8^(th) aspect, the reaction of step (b) is carried out in an aqueoussolution comprising a mixture of water and an organic solvent. Anysuitable organic solvent described above can be used. More specifically,the organic solvent is DMA. In some embodiments, the aqueous solutioncomprises less than 50%, less than 40%, less than 30%, less than 25%,less than 20%, less than 15%, less than 10%, less than 5%, less than 3%,less than 2%, or less than 1% of the organic solvent (e.g. DMA) byvolume.

In some embodiments, for the methods of the present invention describedherein or in the 1^(st), 2^(nd), 3^(rd), 4^(th), 5^(th), 6^(th), 7^(th)or 8^(th) aspect, the modified cytotoxic agent is not purified beforereacting with the cell-binding agent in step (b). Alternatively, themodified cytotoxic agent is purified before reacting with thecell-binding agent in step (b). Any suitable methods described hereincan be used to purify the modified cytotoxic agent. In certainembodiments, the present invention provides isolated modified cytotoxicagent prepared by the present methods. The isolated modified cytotoxicagent can be stored for a period of time before reacting with thecell-binding agent. Preferably, the isolated modified cytotoxic agent isstored under conditions that will prevent the decomposition of themodified cytotoxic agent, for example, as purified solid or as frozensolution or be kept at a low temperature (e.g., less than 10° C. or lessthan 5° C.).

In some embodiments, for the methods of the present invention describedherein or in any one of the embodiments described above, thecell-binding agent-cytotoxic agent conjugate of step (b) is subject to apurification step. In this regard, the cell-binding agent-cytotoxicagent conjugate can be purified from the other components of the mixtureusing tangential flow filtration (TFF), non-adsorptive chromatography,adsorptive chromatography, adsorptive filtration, selectiveprecipitation, or any other suitable purification process, as well ascombinations thereof.

In some embodiments of the invention, the cell-binding agent-cytotoxicagent conjugate is purified using a single purification step (e.g.,TFF). Preferably, the conjugate is purified and exchanged into theappropriate formulation using a single purification step (e.g., TFF). Inother embodiments of the invention, the cell-binding agent cytotoxicagent conjugate is purified using two sequential purification steps. Forexample, the conjugate can be first purified by selective precipitation,adsorptive filtration, absorptive chromatography or non-absorptivechromatography, followed by purification with TFF. One of ordinary skillin the art will appreciate that purification of the cell-bindingagent-cytotoxic agent conjugate enables the isolation of a stableconjugate comprising the cell-binding agent chemically coupled to thecytotoxic agent.

Any suitable TFF systems may be utilized for purification, including aPellicon type system (Millipore, Billerica, Mass.), a Sartocon Cassettesystem (Sartorius AG, Edgewood, N.Y.), and a Centrasette type system(Pall Corp., East Hills, N.Y.)

Any suitable adsorptive chromatography resin may be utilized forpurification. Preferred adsorptive chromatography resins includehydroxyapatite chromatography, hydrophobic charge inductionchromatography (HCIC), hydrophobic interaction chromatography (HIC), ionexchange chromatography, mixed mode ion exchange chromatography,immobilized metal affinity chromatography (IMAC), dye ligandchromatography, affinity chromatography, reversed phase chromatography,and combinations thereof. Examples of suitable hydroxyapatite resinsinclude ceramic hydroxyapatite (CHT Type I and Type II, Bio-RadLaboratories, Hercules, Calif.), HA Ultrogel hydroxyapatite (Pall Corp.,East Hills, N.Y.), and ceramic fluoroapatite (CFT Type I and Type II,Bio-Rad Laboratories, Hercules, Calif.). An example of a suitable HCICresin is MEP Hypercel resin (Pall Corp., East Hills, N.Y.). Examples ofsuitable HIC resins include Butyl-Sepharose, Hexyl-Sepharose,Phenyl-Sepharose, and Octyl Sepharose resins (all from GE Healthcare,Piscataway, N.J.), as well as Macro-prep Methyl and Macro-Prep t-Butylresins (Biorad Laboratories, Hercules, Calif.). Examples of suitable ionexchange resins include SP-Sepharose, CM-Sepharose, and Q-Sepharoseresins (all from GE Healthcare, Piscataway, N.J.), and Unosphere S resin(Bio-Rad Laboratories, Hercules, Calif.). Examples of suitable mixedmode ion exchangers include Bakerbond ABx resin (JT Baker, PhillipsburgN.J.) Examples of suitable IMAC resins include Chelating Sepharose resin(GE Healthcare, Piscataway, N.J.) and Profinity IMAC resin (Bio-RadLaboratories, Hercules, Calif.). Examples of suitable dye ligand resinsinclude Blue Sepharose resin (GE Healthcare, Piscataway, N.J.) andAffi-gel Blue resin (Bio-Rad Laboratories, Hercules, Calif.). Examplesof suitable affinity resins include Protein A Sepharose resin (e.g.,MabSelect, GE Healthcare, Piscataway, N.J.), where the cell-bindingagent is an antibody, and lectin affinity resins, e.g. Lentil LectinSepharose resin (GE Healthcare, Piscataway, N.J.), where thecell-binding agent bears appropriate lectin binding sites. Alternativelyan antibody specific to the cell-binding agent may be used. Such anantibody can be immobilized to, for instance, Sepharose 4 Fast Flowresin (GE Healthcare, Piscataway, N.J.). Examples of suitable reversedphase resins include C4, C8, and C18 resins (Grace Vydac, Hesperia,Calif.).

Any suitable non-adsorptive chromatography resin may be utilized forpurification. Examples of suitable non-adsorptive chromatography resinsinclude, but are not limited to, SEPHADEX™ G-25, G-50, G-100, SEPHACRYL™resins (e.g., S-200 and S-300), SUPERDEX™ resins (e.g., SUPERDEX™ 75 andSUPERDEX™ 200), BIO-GEL® resins (e.g., P-6, P-10, P-30, P-60, andP-100), and others known to those of ordinary skill in the art.

In some embodiments, the purified cell-binding agent-cytotoxic agentconjugate is formulated into a suitable formulation buffer. In someembodiments, the formulation buffer comprises a bisulfite salt, such assodium bisulfite or potassium bisulfite. More specifically, theformulation buffer comprises sodium bisulfite. In some embodiments, theformulation buffer comprises 5 to 200 μM, 10 to 200 μM, 10 to 150 μM, 20to 100 μM, 30 to 90 μM, 40 to 80 μM, 50 to 70 μM, 40 to 60 μM, 45 to 55μM, or 55 to 65 μM of the bisulfite salt (e.g., sodium bisulfite). Inother embodiments, the formulation buffer comprises 20, 30, 40, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 70, 80, 90, or 100 μM of the bisulfite salt (e.g., sodiumbisulfite).

In other embodiments, the formulation buffer further comprisestrehalose. Any suitable amount of trehalose can be used. In someembodiments, the formulation buffer further comprises 2 to 15%, 5 to10%, 6 to 10% or 7 to 9% or 6 to 8% of trehalose by weight.

In another embodiment, the formulation buffer has a pH of 4 to 6, 4 to5, or 4 to 4.5. In other embodiments, the pH for the formulation bufferis 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

In yet other embodiments, the formulation buffer comprises 10 mM sodiumsuccinate, 50 μM sodium bisulfite, 8% trelose dihydrate and 0.01%polysorbate 20 at pH 4.2.

In some embodiments, for the methods of the present invention describedherein, the reaction of step (a) results in no substantial sulfonationof the maleimide group. In some embodiments, less than 50%, 40%, 30%,20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the maleimide groupis sulfonated. The percentage of maleimide sulfonation is equal to thetotal amount of the maleimide-sulfonated cytotoxic agent (the cytotoxicagent having sulfonation on the maleimide only) and the di-sulfonatedcytotoxic agent (the cytotoxic agent having sulfonation on both themaleimide and the imine moieties) divided by the starting amount of theimine-containing cytotoxic agent before its reaction with the bisulfitesalt or the metabisulfite salt.

In a 9^(th) aspect, the present invention provides a method of preparinga cell-binding agent-cytotoxic agent conjugate represented by thefollowing formula:

or a pharmaceutically acceptable salt thereof, comprising the steps of:

(a) reacting an imine-moiety in an imine-containing cytotoxic agentrepresented by the following formula:

or a pharmaceutically acceptable salt thereof, with sulfur dioxide, abisulfite salt or a metabisulfite salt in an aqueous solution at a pH of3.1 to 3.5 to form a modified cytotoxic agent represented by thefollowing formula:

or a pharmaceutically acceptable salt thereof; and

(b) reacting the modified cytotoxic agent or a pharmaceuticallyacceptable salt thereof, with a cell-binding agent Ab to form thecell-binding agent-cytotoxic agent conjugate, wherein:

Ab is an anti-CD123 antibody comprising an immunoglobulin heavy chainhaving the amino acid sequence of SEQ ID NO:25 and an immunoglobulinlight chain having the amino acid sequence of SEQ ID NO:26; and

w is 1 or 2.

In some embodiments, the method of the 9^(th) aspect is carried outunder reaction conditions described in the 1^(st),2^(nd), 3^(rd),4^(th), 5^(th), 6^(th), 7^(th), or 8^(th) aspect and any embodimentsdescribed therein.

In a 10^(th) aspect, the reaction of step (a) in the method of 9^(th)aspect is carried out at a pH of 3.2 to 3.4. More specifically, the pHis 3.3.

In some embodiments, the reaction of step (a) is carried out in thepresence of a buffer solution. Exemplary buffer solutions include, butare not limited to, a citrate buffer, an acetate buffer, a succinatebuffer or a phosphate buffer. More specifically, the buffer is asuccinate buffer.

In a 11^(th) aspect, for the reaction of step (a) in the method of9^(th) aspect, 1.1 to 1.6 equivalents of the bisulfite salt or 0.55 to0.8 equivalents of the metabisulfite salt is reacted with 1 equivalentof the imine-containing cytotoxic agent. The remaining reactionconditions are as described above in the 10^(th) aspect and anyembodiments described therein. In some embodiments, 1.3 to 1.5equivalents of the bisulfite salt or 0.65 to 0.75 equivalents of themetabisulfite salt is reacted with 1 equivalent of the imine-containingcytotoxic agent. More specifically, 1.4 equivalents of the bisulfitesalt or 0.7 equivalents of the metabisulfite salt is reacted with 1equivalent of the imine-containing cytotoxic agent.

In a 12^(th) aspect, the reaction of step (a) in the method of 9^(th)aspect is carried out at a pH of 3.2 to 3.4 and 1.3 to 1.5 equivalentsof the sodium bisulfite salt is reacted with 1 equivalent of theimine-containing cytotoxic agent. In some embodiments, the reaction iscarried out at a pH of 3.3 and 1.4 equivalents of the sodium bisulfitesalt is reacted with 1 equivalent of the imine-containing cytotoxicagent.

In some embodiments, the reaction of step (a) in the method of 9^(th),10^(th), 11^(th) or 12^(th) aspect is carried out in a suitable solventor solvent mixture. In some embodiments, the reaction of step (a) iscarried out in an aqueous solution comprising a mixture of water and anorganic solvent. Any suitable organic solvent described above can beused. More specifically, the organic solvent is DMA. In someembodiments, the aqueous solution comprises less than 50%, less than40%, less than 30%, less than 25%, less than 20%, less than 15%, lessthan 10%, less than 5%, less than 3%, less than 2%, or less than 1% ofthe organic solvent (e.g. DMA) by volume.

In some embodiments, the reaction of step (a) is carried out at asuitable temperature, for example, at room temperature or at 15 to 25°C., for a sufficient period time, such as 1 hour, 2 hours, 3 hours, 4hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours,15 hours, 20 hours, 24 hours, 48 hours, etc.

In some embodiments, the modified cytotoxic agent obtained from thereaction of step (a) is not purified before reacting with thecell-binding agent in step (b).

In some embodiments, the modified cytotoxic agent obtained from thereaction of step (a) is purified before reacting with the cell-bindingagent in step (b). Any suitable purification methods described hereincan be used.

In a 13^(th) aspect, the reaction of step (b) in the method of 9^(th)aspect is carried out at a pH of 5.5 to 6.5; and the remaining reactionconditions are as described in the 9^(th), 10^(th), 11^(th) or 12^(th)aspect. In some embodiments, the pH is 5.5, 5.6, 5.7, 5.8, 5.9, 6.0,6.1, 6.2, 6.3, 6.4 or 6.5. In some embodiments, the pH is 6.0.

In some embodiments, the conjugate prepared by the methods of the9^(th), 10^(th), 11^(th), 12^(th) or 13^(th) aspect is purified by asuitable method described herein. In one embodiment, the conjugate ispurified by tangential flow filtration to yield purified conjugate.

In some embodiments, the purified conjugate is formulated in aformulation buffer comprising 40 to 80 μM of sodium bisulfite having apH of 4 to 5. In some embodiments, the formulation buffer comprises 50μM of sodium bisulfite having a pH of 4.2. In some embodiments, theformulation buffer comprises 10 mM sodium succinate, 50 μM sodiumbisulfite, 8% trelose dihydrate and 0.01% polysorbate 20 at pH 4.2.

In some embodiments, for the methods of the present invention describedherein (e.g., the 1^(st), 2^(th), 3^(rd), 4^(th), 5^(th), 6^(th), 7^(th)or 8^(th) aspect and any embodiments described therein), thecell-binding agent is not an anti-CD123 antibody.

In some embodiments, for methods of the present invention describedherein (e.g., the 1^(st)2^(nd), 3^(rd), 4^(th), 5^(th), 6^(th), 7^(th)or 8^(th) aspect and any embodiments described therein), thecell-binding agent is not an anti-CD123 antibody comprising animmunoglobulin heavy chain having the amino acid sequence of SEQ IDNO:25 and an immunoglobulin light chain having the amino acid sequenceof SEQ ID NO:26.

In some embodiments, the cell-binding agent-cytotoxic agent conjugateprepared by the methods of the present invention described herein (e.g.,the 1^(st), 2^(nd), 3^(rd), 4^(th), 5^(th), 6^(th), 7^(th) or 8^(th)aspect and any embodiments described therein) is not a conjugaterepresented by the following structure:

or a pharmaceutically acceptable salt thereof, wherein Ab is ananti-CD123 antibody comprising an immunoglobulin heavy chain having theamino acid sequence of SEQ ID NO:25 and an immunoglobulin light chainhaving the amino acid sequence of SEQ ID NO:26; and

w is 1 or 2.

Imine-Containing Cytotoxic Agents and Conjugates of the PresentInvention

In some embodiments, for the methods of the present invention describedherein, the imine-containing cytotoxic agent is represented by thefollowing formula:

or a pharmaceutically acceptable salt thereof, and

the cell-binding agent-cytotoxic agent conjugate is represented by thefollowing formula:

wherein D^(M) is the modified cytotoxic agent comprising the modifiedimine moiety represented by the following formula:

or a pharmaceutically acceptable salt thereof; L is a linker;

represents the cell-binding agent linked to the cytotoxic agent via athiol group; and w is an integer from 1 to 10.

In some embodiments, the CBA is an antibody and the antibody is linkedto the cytotoxic agent via one or more cysteine thiol group. In someembodiments, the free thiol group is on an engineered Cys residue in theheavy chain CH3 region of an antibody, at the EU/OU numbering position442 of that heavy chain (or C442 for short). More specifically, thecysteine residue at position 442 is recombinantly introduced into theantibody.

In other embodiments, w is 1 or 2. More specifically, w is 2.

The following describes certain embodiments and specific embodiments forthe methods of the present invention described herein (e.g., the methodsof the 1^(st), 2^(nd), 3^(rd), 4^(th), 5^(th), 6^(th), 7^(th) or 8^(th)aspect described above and embodiments described therein).

In a 1^(st) specific aspect, for the imine-containing cytotoxic agent offormula (A) or the conjugate of formula (B), D is represented by thefollowing structural formula:

or a pharmaceutically acceptable salt thereof, wherein:

one of L′, L″, and L′″ is represented by the following formula:

—Z₁—P₁—Z₂—R_(x1)—C(═O)—  (A′), or

—N(R^(e))—R_(x1)—C(═O)—  (D′);

and the other two are each independently selected from —H, an optionallysubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl havingfrom 1 to 10 carbon atoms, a polyethylene glycol unit—(CH₂CH₂O)_(n)—R^(c), halogen, guanidinium [—NH(C═NH)NH₂], —OR, —NR′R″,—NO₂, —NR′COR″, —SR, —SOR′, —SO₂R′, —SO₃H, —OSO₃H, —SO₂NR′R″, cyano, anazido, —COR′, —OCOR′, and —OCONR′R″;

one of the Z₁ and Z₂ is —C(═O)—, and the other is —NR₅—;

P₁ is an amino acid residue or a peptide containing between 2 to 20amino acid residues;

R_(x1) is an optionally substituted linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms;

R^(e) is —H, a linear, branched or cyclic alkyl, alkenyl or alkynylhaving 1 to 10 carbon atoms or —(CH₂—CH₂—O)_(n)—R^(k), wherein R^(k) isa —H, a linear, branched cyclic alkyl having 1 to 6 carbon atoms,optionally bearing a secondary amino (e.g., —NHR¹⁰¹) or tertiary amino(—NR¹⁰¹R¹⁰²) group or a 5- or 6-membered nitrogen containingheterocycle, such as piperidine or morpholine, wherein R¹⁰¹ and R¹⁰² areeach independently a linear, branched, or cyclic alkyl, alkenyl oralkynyl having 1 to 10 carbon atoms;

R, for each occurrence, is independently selected from the groupconsisting of —H, an optionally substituted linear, branched or cyclicalkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, apolyethylene glycol unit —(CH₂CH₂O)_(n)—R^(c), an optionally substitutedaryl having 6 to 18 carbon atoms, an optionally substituted 5- to18-membered heteroaryl ring containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur, or anoptionally substituted 3- to 18-membered heterocyclic ring containing 1to 6 heteroatoms independently selected from O, S, N and P;

R′ and R″ are each independently selected from —H, —OH, —OR, —NHR, —NR₂,—COR, an optionally substituted linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethyleneglycol unit —(CH₂CH₂O)_(n)—R^(c), and an optionally substituted 3- to18-membered heterocyclic ring having 1 to 6 heteroatoms independentlyselected from O, S, N and P;

R^(c) is —H or an optionally substituted linear or branched alkyl having1 to 4 carbon atoms;

n is an integer from 1 to 24;

X₁′ is selected from —H, an amine-protecting group, an optionallysubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl havingfrom 1 to 10 carbon atoms, a polyethylene glycol unit—(CH₂CH₂O)_(n)—R^(c), an optionally substituted aryl having 6 to 18carbon atoms, an optionally substituted 5- to 18-membered heteroarylring containing one or more heteroatoms independently selected fromnitrogen, oxygen, and sulfur, and an optionally substituted 3- to18-membered heterocyclic ring containing 1 to 6 heteroatomsindependently selected from O, S, N and P;

Y₁′ is selected from —H, an oxo group (i.e., Y₁′ together with thecarbon atom to which it is attached form the —C(═O)— group), anoptionally substituted linear, branched or cyclic alkyl, alkenyl oralkynyl having from 1 to 10 carbon atoms, an optionally substituted 6-to 18-membered aryl, an optionally substituted 5- to 18-memberedheteroaryl ring containing one or more heteroatoms independentlyselected from nitrogen, oxygen, and sulfur, an optionally substituted 3-to 18-membered heterocyclic ring having 1 to 6 heteroatoms;

R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′ and R₄′ are each independently selectedfrom the group consisting of —H, an optionally substituted linear,branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbonatoms, a polyethylene glycol unit —(CH₂CH₂O)_(n)—R^(c), halogen,guanidinium [—NH(C═NH)NH₂], —OR, —NR′R″, —NO₂, —NCO, —NR′COR″, —SR,—SOR′, —SO₂R′, —SO₃ ⁻H, —OSO₃H, —SO₂NR′R″, cyano, an azido, —COR′,—OCOR′, and —OCONR′R″;

R₆ is —H, —R, —OR, —SR, —NR′R″, —NO₂, or halogen;

G is —CH— or —N—;

A and A′ are the same or different, and are independently selected from—O—, oxo (—C(═O)—), —CRR′O—, —CRR′—, —S—, —CRR'S—, —NR₅ and —CRR′N(R₅)—;and

R₅ for each occurrence is independently —H or an optionally substitutedlinear or branched alkyl having 1 to 10 carbon atoms.

In a more specific embodiment, D is represented by formula (IGN1′) or(IGN1).

In another more specific embodiment, for formulas (IGN1′)-(IGN4′) and(IGN1)-(IGN4), one of L′, L″ and L′″ is represented by formula (A′) or(D′), and the others are —H, an linear or branched alkyl having from 1to 6 carbon atoms, halogen, —OH, (C₁-C₆)alkoxy, or —NO₂.

In another more specific embodiment, for formulas (IGN1′)-(IGN4′) and(IGN1)-(IGN4), L′ is represented by formula (A′); and L″ and L′″ areboth —H.

In another more specific embodiment, for formulas (IGN1′)-(IGN4′) and(IGN1)-(IGN4), L′ is represented by formula (D′); and L″ and L′″ areboth —H.

In another more specific embodiment, for formulas (IGN1′)-(IGN4′) and(IGN1)-(IGN4), R_(x1) is a linear, branched or cyclic alkyl having 1 to6 carbon atoms optionally substituted with halogen, —OH, —SO₃H,(C₁-C₃)alkyl, (C₁-C₃)alkoxy, halo(C₁-C₃)alkyl, or a charged substituentor an ionizable group Q.

In a 2^(nd) specific aspect, for formulas (IGN1′)-(IGN4′) and(IGN1)-(IGN4), L′ is represented by the following formula:

—NR₅—P₁—C(═O)—(CR_(a)R_(b))_(s)—C(═O)—  (B1′);

—NR₅—P₁—C(═O)—Cy-(CR_(a)R_(b))_(s1′)—C(═O)—  (B2′);

—C(═O)—P₁—NR₅—(CR_(a)R_(b))_(s)—C(═O)—  (C1′), or

—C(═O)—P₁—NR₅-Cy-(CR_(a)R_(b))_(s1′)—C(═O)—  (C₂′)

wherein:

R_(a) and R_(b), for each occurrence, are each independently —H,(C₁-C₃)alkyl or a charged substituent or an ionizable group Q;

s is an integer from 1 to 6;

s1′ is 0 or an integer from 1 to 6; and

Cy is a cyclic alkyl having 5 or 6 ring carbon atoms optionallysubstituted with halogen, —OH, (C₁-C₃)alkyl, (C₁-C₃)alkoxy, orhalo(C₁-C₃)alkyl;

and the remaining variables are as described above in the 1^(st)specific aspect or any specific or more specific embodiments describedtherein.

In a more specific embodiment, R_(a) and R_(b) are both H; Cy informulas (B2′) and (C2′) is cyclohexane; and R₅ is H or Me. Even morespecifically, s1′ is 0 or 1.

In a 3^(rd) specific aspect, for formulas (IGN1′)-(IGN4′) and(IGN1)-(IGN4), P₁ is a peptide containing 2 to 10 amino acid residues;and the remaining variables are as described in the 1^(st) or 2^(nd)specific aspect or any specific or more specific embodiments describedtherein.

In specific embodiments, P₁ is a peptide containing 2 to 5 amino acidresidues.

In another specific embodiments, P₁ is Gly-Gly-Gly, Ala-Val, Val-Cit,Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit,Phe-Ala, Phe-N⁹-tosyl-Arg, Phe-N⁹-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys,Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2), Gly-Phe-Leu-Gly (SEQ ID NO:3), Val-Arg, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit,D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg,D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met,Gln-Val, Asn-Ala, Gln-Phe and Gln-Ala. Even more specifically, P isGly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

In a 4^(th) specific aspect, for formulas (IGN1′)-(IGN4′) and(IGN1)-(IGN4), X₁′ is selected from the group consisting of —H, —OH, anoptionally substituted linear, branched or cyclic alkyl, alkenyl oralkynyl having from 1 to 10 carbon atoms, and phenyl; Y₁′ is selectedfrom the group consisting of —H, an oxo group, an optionally substitutedlinear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10carbon atoms; and the remaining variables are as described above in the1^(st), 2^(nd), or 3^(rd) specific aspect or any specific or morespecific embodiments described therein. More specifically, X₁′ is —H,—OH, (C₁-C₃)alkyl, halo(C₁-C₃)alkyl, or phenyl; and Y₁′ is —H, an oxogroup, (C₁-C₃)alkyl or halo(C₁-C₃)alkyl. In another more specificembodiment, X₁′ is —H, —OH or -Me; and Y₁′ is —H or oxo. Even morespecifically, X₁′ is —H; and Y₁′ is —H.

In a 5^(th) specific aspect, for formulas (IGN1′)-(IGN4′) and(IGN1)-(IGN4), A and A′ are the same or different, and are —O—, —S—,—NR₅—, or oxo —(C═O)—; and the remaining variables are as describedabove in the 1^(st), 2^(nd)3^(rd) or 4^(th) specific aspect or anyspecific or more specific embodiments described therein. Morespecifically, A and A′ are the same or different, and are —O— or —S—.Even more specifically, A and A′ are —O—.

In a 6^(th) specific aspect, for formulas (IGN1′)-(IGN4′) and(IGN1)-(IGN4), R₆ is —OR, wherein R is an alkyl group having 1 to 6carbon atoms; and the remaining variables are as described above in the1^(st), 2^(nd)3^(rd)4^(th) or 5^(th) specific aspect or any specific ormore specific embodiments described therein. More specifically, R₆ is—OMe.

In a 7^(th) specific aspect, for formulas (IGN1)-(IGN4), R₁, R₂, R₃, R₄,R₁′, R₂′, R₃′ and R₄′ are each independently —H, halogen, —NO₂, —OH,(C₁-C₃)alkyl, halo(C₁-C₃)alkyl or (C₁-C₃)alkoxy; and the remainingvariables are as described above in the 1^(st), 2^(nd)3^(rd)4^(th),5^(th) or 6^(th) specific aspect or any specific or more specificembodiments described therein. More specifically, R₁, R₂, R₃, R₄, R₁′,R₂′, R₃′ and R₄′ are all —H.

In a 8^(th) specific aspect, for formulas (IGN1′)-(IGN4′) and(IGN1)-(IGN4), R, R′, R″ and R₅ are each independently —H or(C₁-C₃)alkyl; and the remaining variables are as described above in the1^(st), 2^(nd), 3^(rd), 4^(th), 5^(th), 6^(th) or 7^(th) specific aspector any specific or more specific embodiments described therein.

In a 9^(th) specific aspect, for formulas (IGN1′)-(IGN4′) and(IGN1)-(IGN4):

R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′ and R₄′ are all —H;

R₆ is —OMe;

X₁′ and Y₁′ are both —H; and

A and A′ are —O—;

and the remaining variables are as described above in the 1^(st),2^(nd), or 3^(rd) specific aspect or any specific or more specificembodiments described therein. More specifically, R, R′, R″ and R₅ areeach independently —H or (C₁-C₃)alkyl. Even more specifically, R, R′, R″and R₅ are all —H.

In a 10^(th) specific aspect, for the imine-containing cytotoxic agentof formula (A) or the conjugate of formula (B), D is represented by thefollowing structural formula:

or a pharmaceutically acceptable salt thereof; and the remainingvariables are described for formula (A) or (B) described above.

In a 11^(th) specific aspect, for the imine-containing cytotoxic agentof formula (A) or the conjugate of formula (B), -L- is represented bythe following structural formula:

wherein:

-   -   s1 is the site covalently linked to D; s2 is the site covalently        linked to the maleimide group;

R₂₃ and R₂₄, for each occurrence, are independently H or an optionallysubstituted alkyl;

m′ is an integer between 0 and 10; and

R^(h′) is H or an optionally substituted alkyl;

and the remaining variables are as described above for formula (A) orformula (B), or in the 1^(st) to 10^(th) specific aspect, or anyspecific or more specific embodiments described therein.

In a specific embodiment, m′ is an integer from 1 to 6. Even morespecifically, m′ is an integer from 1 to 3.

In another specific embodiment, R₂₃ and R₂₄, for each occurrence, areindependently H or a (C₁-C₃)alkyl. Even more specifically, R₂₃ and R₂₄are both H.

In another specific embodiment, R^(h)′ is H or a (C₁-C₃)alkyl. Morespecifically, R^(h)′ is H.

In another specific embodiment, R₂₃ and R₂₄ are both H; m′ is an integerfrom 1 to 6.

Even more specifically, m′ is an integer from 1 to 3.

In a 12^(th) specific aspect, for the imine-containing cytotoxic agentof formula (A) or the conjugate of formula (B), -L- is represented bythe following structural formula:

wherein s1 is the site covalently linked to D; s2 is the site covalentlylinked to the maleimide group; and the remaining variables are asdescribed above for formula (A) or formula (B), or in the 1^(st) to10^(th) specific aspect, or any specific or more specific embodimentsdescribed therein.

In a 13^(th) specific aspect, the imine-containing cytotoxic agent isrepresented by the following formula:

or a pharmaceutically acceptable salt thereof, and the modifiedcytotoxic agent is represented by the following formula:

or a pharmaceutically acceptable salt thereof. More specifically, themodified cytotoxic agent is represented by the following formula:

or a sodium or potassium salt thereof. Even more specifically, themodified cytotoxic agent is represented by the following formula:

In some embodiments, the imine-containing cytotoxic agent is representedby the following formula:

or a pharmaceutically acceptable salt thereof, and the modifiedcytotoxic agent is represented by the following formula:

or a pharmaceutically acceptable salt thereof. More specifically, themodified cytotoxic agent is represented by the following formula:

or a sodium or potassium salt thereof. Even more specifically, themodified cytotoxic agent is represented by the following formula:

In a 14^(th) specific aspect, for the imine-containing cytotoxic agentof formula (A) or the conjugate of formula (B), D is represented by thefollowing structural formula:

or a pharmaceutically acceptable salt thereof, wherein:

X′ is selected from the group consisting of —H, —OH, a substituted orunsubstituted linear, branched or cyclic alkyl, alkenyl or alkynylhaving from 1 to 10 carbon atoms, phenyl, and an amine-protecting group;

Y′ is selected from the group consisting of —H, an oxo group, asubstituted or unsubstituted linear, branched or cyclic alkyl, alkenylor alkynyl having from 1 to 10 carbon atoms;

A and A′ are selected from —O— and —S—;

W′ is absent, or selected from —O—, —N(R^(e))—, —N(R^(e))—C(═O)—,—N(C(═O)R^(e))—, —S— or —CH₂—S—, —CH₂NR^(e)—;

R^(x) is absent or selected from a linear, branched or cyclic alkylhaving 1 to 10 carbon atoms;

R^(e) is —H, a linear, branched or cyclic alkyl, alkenyl or alkynylhaving 1 to 10 carbon atoms or —(CH₂—CH₂—O)_(n)—R^(k), wherein R^(k) isa —H, a linear, branched cyclic alkyl having 1 to 6 carbon atoms,optionally bearing a secondary amino (e.g., —NHR¹⁰¹) or tertiary amino(—NR¹⁰¹R¹⁰²) group or a 5- or 6-membered nitrogen containingheterocycle, wherein R¹⁰¹ and R¹⁰² are each independently a linear,branched, or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbonatoms;

n is an integer from 1 to 24;

G is selected from —CH— or —N—;

R₆ is —H, —R, —OR, —SR, —NR′R″, —NO₂, or halogen; and

R is —H, an optionally substituted linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms or a PEG group—(CH₂CH₂O)_(n)—R^(c), wherein n is an integer from 1 to 24, and R^(c) isa linear or branched alkyl having 1 to 4 carbon atoms;

R′ and R″ are each independently selected from —H, —OH, —OR, —NRR^(g′),—COR, an optionally substituted linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms, an optionallysubstituted aryl having from 6 to 18 carbon atoms, an optionallysubstituted 3- to 18-membered heterocyclic ring having 1 to 6heteroatoms selected from O, S, N and P, a PEG group—(CH₂CH₂O)_(n)—R^(c), and R^(g′) is —H, an optionally substitutedlinear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10carbon atoms or a PEG group —(CH₂CH₂O)_(n)—R^(c).

In a more specific embodiment, D is represented by formula (IGN5′) or(IGN5) above, or a pharmaceutically acceptable salt thereof.

In another specific embodiment, for formulas (IGN5′)-(IGN8′) and(IGN5)-(IGN8):

-   -   X′ and Y′ are both —H;    -   A and A′ are both —O—;    -   R₆ is —OMe;    -   W′ is —N(R^(e))— or —N(R′)—C(═O)—;    -   R^(e) is —H, a linear or branched alkyl having 1 to 4 carbon        atoms or —(CH₂—CH₂—O)_(n)—R^(k), wherein R^(k) is a —H, a linear        or branched alkyl having 1 to 4 carbon atoms;    -   n is an integer from 2 to 6; and    -   R^(x) is a linear or branched alkyl having 1 to 6 carbon atoms.

In a 15^(th) specific aspect, for the imine-containing cytotoxic agentof formula (A) or the conjugate of formula (B), D is represented by thefollowing structural formula:

or a pharmaceutically acceptable salt thereof, wherein the remainingvariables are as described above for formula (A) or (B).

In a 16^(th) specific aspect, for the imine-containing cytotoxic agentof formula (A) or the conjugate of formula (B), -L- is represented bythe following structural formula:

wherein:

s1 is the site covalently linked to D, and s2 is the site covalentlylinked to the maleimide group;

E is —(CR₁₀R₁₁)_(q)—, cycloalkyl, or cycloalkylalkyl;

Z is absent, —SO₂NR₉—, —NR₉SO₂—, —C(═O)—NR₉—, —NR₉—C(═O)—, —C(═O)—O—,—O—C(═O)—, —C(═O)—NR₉—(CH₂CH₂O)_(p)—, —NR₉—C(═O)—(CH₂CH₂O)_(p)—,—(OCH₂CH₂)_(p)—C(═O)NR₉—, or —(OCH₂CH₂)_(p)—NR₉—C(═O)—;

p is an integer from 1 to 24;

Q is H, a charged substituent, or an ionizable group;

R₉, R₁₀, R₁₁, R₁₂, and R₁₃, for each occurrence, are independently H oran optionally substituted alkyl; and,

q and r, for each occurrence, are independently an integer between 0 and10; and the remaining variables are as described above for formula (A)or formula (B), or in 14^(th) or 15^(th) specific aspect or any specificor more specific embodiments described therein.

In a more specific embodiment, q and r, are independently an integerfrom 1 to 6, more specifically, 1 to 3.

In another more specific embodiment, R₉, R₁₀, R₁₁, R₁₂, and R₁₃, foreach occurrence, are independently H or a C₁₋₃alkyl. More specifically,R₉, R₁₀, R₁₁, R₁₂, and R₁₃ are all H.

In another more specific embodiment, p is an integer from 2 to 14. Morespecifically, p is an integer from 2 to 8, 2 to 6 or 2 to 4.

In a more specific embodiment, E is —(CR₁₀R₁₁)_(q)—; and the remainingvariables in formula (L2) are as described above in the 16^(th) specificaspect.

In another more specific embodiment, E is

and the remaining variables in formula (L2) are as described above inthe 16^(th) specific aspect.

In yet another specific embodiment, Z is —C(═O)—NR₉— or —NR₉—C(═O)—; andthe remaining variables in formula (L2) are as described above in the16^(th) specific aspect or any specific or more specific embodimentsdescribed above. Even more specifically, R₉ is H or Me. Alternatively,R₉ is H.

In yet another more specific embodiment, Q is i) H; ii) —SO₃H, —Z′—SO₃H,—OPO₃H₂, —Z′—OPO₃H₂, —PO₃H₂, —Z′—PO₃H₂, —CO₂H, —Z′—CO₂H, —NR₁₄R₁₅, or—Z′—NR₁₄R₁₅, or a pharmaceutically acceptable salt thereof; or, iii)—N⁺R₁₄R₁₅R₁₆X⁻ or —Z′—NR₁₄R₁₅R₁₆X⁻; Z′ is an optionally substitutedalkylene, an optionally substituted cycloalkylene or an optionallysubstituted phenylene; R₁₄ to R₁₆ are each independently an optionallysubstituted alkyl; and X⁻ is a pharmaceutically acceptable anion; andthe remaining variables in formula (L2) are as described above in the16^(th) specific embodiment or any more specific embodiments describedabove. In some embodiments, Z′ is an optionally substituted alkylene. Inyet other embodiments, Z′ is a C₁₋₃alkylene (e.g., —CH₂); and R₁₄ to R₁₆are each independently a C₁₋₄alkyl.

In yet another ore specific embodiment, Q is H, or —SO₃H, or apharmaceutically acceptable cation (e.g., sodium or potassium salt); andthe remaining variables in formula (L2) are as described above in the16^(th) specific aspect or any specific or more specific embodimentsdescribed above.

In another more specific embodiment, for formula (L2):

R₁₂ and R₁₃, for each occurrence, are each independently H or(C₁-C₃)alkyl;

Q is H or —SO₃H or a pharmaceutically acceptable salt thereof

Z is —C(═O)—NR₉— or —NR₉—C(═O)—;

R₉ is H or (C₁-C₃)alkyl;

E is —(CR₁₀R₁₁)_(q)—.

R₁₀ and R₁₁, for each occurrence, are independently H or (C₁-C₃)alkyl;and

q and r are each an integer from 1 to 5.

In a 17^(th) specific aspect, for the imine-containing cytotoxic agentof formula (A) or the conjugate of formula (B), -L- is represented byany one of the following structural formulae:

or a pharmaceutically acceptable salt (e.g., sodium or potassium salt)thereof, wherein s1 is the site covalently linked to D, and s2 is thesite covalently linked to the maleimide group; q and r are eachindependently an integer from 1 to 6; and the remaining variables are asdescribed above for formula (A) or formula (B), or in 14^(th) or 15^(th)specific aspect or any specific or more specific embodiments describedtherein. More specifically, q and r are each independently an integerfrom 1 to 3.

In a 18^(th) specific aspect, for the imine-containing cytotoxic agentof formula (A) or the conjugate of formula (B), -L- is represented byany one of the following structural formulae:

or a pharmaceutically acceptable salt (e.g., sodium or potassium salt)thereof, wherein s1 is the site covalently linked to D, and s2 is thesite covalently linked to the maleimide group; and the remainingvariables are as described above for formula (A) or formula (B), or in14^(th) or 15^(th) specific aspect or any specific or more specificembodiments described therein.

In a 19^(th) specific aspect, for the imine-containing cytotoxic agentof formula (A) or the conjugate of formula (B), -L- is represented bythe following structural formula:

wherein:

s1 is the site covalently linked to D; s2 is the site covalently linkedto the maleimide group;

R₁₉, R₂₀, R₂₁ and R₂₂, for each occurrence, are independently H or anoptionally substituted alkyl;

m and n are each independently an integer between 0 and 10;

R^(h) is H or an optionally substituted alkyl;

P_(L) is an optionally substituted alkylene, —(CH₂—CH₂—O)_(j)— (whereinthe oxygen atom is connected to the —(C═O)— group connected to P), anamino acid residue or a peptide containing 2 to 20 amino acid residues;and

j is an integer from 1 to 24; and the remaining variables are asdescribed above for formula (A) or formula (B), or the 14^(th) or15^(th) specific aspect, or any specific or more specific embodimentsdescribed therein.

In a 20^(th) specific aspect, for formula (L3), R₁₉, R₂₀, R₂₁ and R₂₂are each H or a (C₁-C₃)alkyl; m and n are each independently an integerbetween 1 and 6; and the remaining variables are as described above inthe 19^(th) specific embodiment or any specific or more specificembodiment described therein. More specifically, R₁₉, R₂₀, R₂₁ and R₂₂are all H. Even more specifically, R₁₉, R₂₀, R₂₁ and R₂₂ are all H; andm and n are each independently an integer between 1 and 3.

In a 21^(st) specific aspect, for formula (L3), P_(L) is an amino acidresidue or a peptide containing 2 to 10 amino acid residues; and theremaining variables are as described above in the 19^(th) or 20^(th)specific aspect or any specific or more specific embodiment describedtherein. More specifically, P_(L) is a peptide containing 2 to 5 aminoacid residues.

In some embodiments, each amino acid residue is the residue of an aminoacid independently selected from: a naturally occurring amino acid, asynthetic amino acid, an amino acid analog, and an amino acid mimeticthat functions in a manner similar to the naturally occurring aminoacids.

In other embodiments, each amino acid residue is the residue of an aminoacid independently selected from the group consisting of: Histidine,Alanine, Isoleucine, Arginine, Leucine, Asparagine, Lysine, Asparticacid, Methionine, Cysteine, Phenylalanine, Glutamic acid, Threonine,Glutamine, Tryptophan, Glycine, Valine, Proline, Serine, Tyrosine,N-methyl-Histidine, N-methyl-Alanine, N-methyl-Isoleucine,N-methyl-Arginine, N-methyl-Leucine, N-methyl-Asparagine,N-methyl-Lysine, N-methyl-Aspartic acid, N-methyl-Methionine,N-methyl-Cysteine, N-methyl-Phenylalanine, N-methyl-Glutamic acid,N-methyl-Threonine, N-methyl-Glutamine, N-methyl-Tryptophan,N-methyl-Glycine, N-methyl-Valine, N-methyl-Proline, N-methyl-Serine,N-methyl-Tyrosine, hydroxyproline, γ-carboxyglutamate, selinocysteine,O-phosphoserine, homoserine, norleucine, methionine sulfoxide,methionine methyl sulfonium, citrulline, Ornithine, cysteine sulfonicacid, cysteine sulfinic acid, 3-aminoalanine, 3-dimethylaminoalanine,2-amino-4-(dimethylamino)butanoic acid, 2,4-diaminobutanoic acid,2-amino-6-(dimethylamino)hexanoic acid,2-amino-5-(dimethylamino)pentanoic acid, and β-alanine, eachindependently as an L or D isomer. More specifically, each amino acidresidue is the residue of an independently selected glycine or alanine.

In other embodiments, P_(L) is a peptide cleavable by a protease. Morespecifically, P_(L) is a peptide cleavable by a protease expressed intumor tissue. Alternatively, P_(L) is a peptide cleavable by a lysosomalprotease.

In yet other embodiments, P_(L) is selected from the group consistingof: Ala-Val, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit,Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Phe-N⁹-tosyl-Arg, Phe-N⁹-nitro-Arg,Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu,Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ IDNO: 2), Gly-Phe-Leu-Gly (SEQ ID NO: 3), Val-Arg, Arg-Arg, Val-D-Cit,Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit,D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala,D-Ala-D-Ala, Ala-Met, Met-Ala, Gln-Val, Asn-Ala, Gln-Phe, Gln-Ala,Gly-Gly-Gly, Ala-Ala-Ala, D-Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-D-Ala,Ala-Val-Cit, Ala-Val-Ala, and β-Ala-Gly-Gly-Gly. More specifically,P_(L) is Gly-Gly-Gly, Ala-Ala-Ala, D-Ala-Ala-Ala, Ala-D-Ala-Ala,Ala-Val-Ala, or β-Ala-Gly-Gly-Gly.

In a 22^(nd) specific aspect, for the imine-containing cytotoxic agentof formula (A) or the conjugate of formula (B), -L- is represented bythe following structural formula:

or a pharmaceutically acceptable salt thereof, wherein s1 is the sitecovalently linked to D; s2 is the site covalently linked to themaleimide group; and the remaining variables are as described above forformula (A) or formula (B), or in the 14^(th) or 15^(th) specificaspect.

In a 23^(rd) specific aspect, the imine-containing cytotoxic agent isrepresented by the following formula:

or a pharmaceutically acceptable salt thereof, and the modifiedcytotoxic agent is represented by the following formula:

or a pharmaceutically acceptable salt thereof.

In amore specific embodiment, the modified cytotoxic agent isrepresented by the following formula:

or a sodium or potassium salt thereof. Even more specifically, themodified cytotoxic agent is represented by the following formula:

In some embodiments, the imine-containing cytotoxic agent is representedby the following formula:

or a pharmaceutically acceptable salt thereof, and the modifiedcytotoxic agent is represented by the following formula:

or a pharmaceutically acceptable salt thereof.

In a 24^(th) specific aspect, the conjugate that can be prepared by themethods of the present invention is represented by the followingstructural formula:

or a pharmaceutically acceptable salt (e.g., sodium or potassium salt)thereof.

In one aspect, for the imine-containing cytotoxic agent of formula (A)or the conjugate of formula (B), D is an imine-containing PBD compound.

In a 25^(th) specific aspect, for the imine-containing cytotoxic agentof formula (A) or the conjugate of formula (B), D is represented by thefollowing formula:

or a pharmaceutically acceptable salt thereof, wherein:

W is selected from C═O, C═S, CH₂, BH, SO and SO₂;

X₁′ is selected from —H, an amine-protecting group, an optionallysubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl havingfrom 1 to 10 carbon atoms, a polyethylene glycol unit—(CH₂CH₂O)_(n)—R^(c), an optionally substituted aryl having 6 to 18carbon atoms, an optionally substituted 5- to 18-membered heteroarylring containing one or more heteroatoms independently selected fromnitrogen, oxygen, and sulfur, and an optionally substituted 3- to18-membered heterocyclic ring containing 1 to 6 heteroatomsindependently selected from O, S, N and P;

Y₁′ is selected from —H, an oxo group, an optionally substituted linear,branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbonatoms, an optionally substituted 6- to 18-membered aryl, an optionallysubstituted 5- to 18-membered heteroaryl ring containing one or moreheteroatoms independently selected from nitrogen, oxygen, and sulfur, anoptionally substituted 3- to 18-membered heterocyclic ring having 1 to 6heteroatoms;

R₆ is —H, —R, —OR, —SR, —NR′R″, —NO₂, or halogen;

A and A′ are the same or different, and are independently selected from—O—, oxo (—C(═O)—), —CRR′O—, —CRR′—, —S—, —CRR'S—, —NR₅ and —CRR′N(R₅)—,

R, for each occurrence, is independently selected from the groupconsisting of —H, an optionally substituted linear, branched or cyclicalkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, apolyethylene glycol unit —(CH₂CH₂O)_(n)—R^(c), an optionally substitutedaryl having 6 to 18 carbon atoms, an optionally substituted 5- to18-membered heteroaryl ring containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur, or anoptionally substituted 3- to 18-membered heterocyclic ring containing 1to 6 heteroatoms independently selected from O, S, N and P;

R′ and R″ are each independently selected from —H, —OH, —OR, —NHR, —NR₂,—COR, an optionally substituted linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethyleneglycol unit —(CH₂CH₂O)_(n)—R^(c), and an optionally substituted 3- to18-membered heterocyclic ring having 1 to 6 heteroatoms independentlyselected from O, S, N and P;

R^(c) is —H or a substituted or unsubstituted linear or branched alkylhaving 1 to 4 carbon atoms, or the linking group with the reactive groupbonded thereto;

n is an integer from 1 to 24;

R₅ for each occurrence is independently —H or an optionally substitutedlinear or branched alkyl having 1 to 10 carbon atoms;

D₀ and D₀′ are the same or different, and are independently absent orselected from the group consisting of an optionally substituted linear,branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbonatoms, an amino acid, a peptide bearing 2 to 6 amino acids, and apolyethylene glycol unit (—OC₂CH₂);

L is absent, a linker, a polyethylene glycol unit (—OCH₂CH₂)_(n)—, anoptionally substituted linear, branched or cyclic alkyl or alkenylhaving 1 to 10 carbon atoms, an optionally substituted phenyl group, anoptionally substituted 3 to 18-membered heterocyclic ring or a 5- to18-membered heteroaryl ring having 1 to 6 heteroatoms independentlyselected from O, S, N and P;

R_(a), R_(a)′, R_(b) and R_(b)′ are the same or different, and areindependently selected from the group consisting of —H, halide, or anoptionally substituted branched, linear or cyclic alkyl having 1 to 10carbon atoms; or R_(a) and R_(a)′ and/or R_(b) and R_(b)′ together forma double bond containing group ═B and ═B′ respectively;

═B and ═B′ are the same or different and independently selected from anoptionally substituted branched or linear alkenyl or a carbonyl group;

Q is Q₁-Ar-Q₂;

Q′ is Q₁′—Ar′-Q₂′;

Q₁ and Q₁′ are each independently absent, a linear, branched or cyclicalkyl from 1 to 6 carbon atoms or a —CH═CH unit;

Ar and Ar′ are each independently absent, or represent an aryl group;

Q₂ and Q₂′ are each independently selected from —H, a linker, asubstituted or unsubstituted linear, branched or cyclic alkyl, alkenylor alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit—R^(c′)—(OCH₂CH₂)_(n)—R^(c), or a substituent selected from a halogen,guanidinium [—NH(C═NH)NH₂], —R, —OR, —NR′R″, —NO₂, —NCO, —NR′COR″,—NR′(C═O)OR″, —SR, a sulfoxide represented by —SOR′, a sulfonerepresented by —SO₂R′, a sulfonate —SO₃M, a sulfate —OSO₃M, asulfonamide represented by SO₂NR′R″, cyano, an azido, —COR′, —OCOR′ or—OCONR′R″; and

R^(c′) is absent or selected from linear or branched alkyl, alkenyl oralkynyl having 1 to 5 carbon atoms.

In a 26^(th) specific aspect, for the imine-containing cytotoxic agentof formula (A) or the conjugate of formula (B), D is selected from oneof the following:

or a pharmaceutically acceptable salt thereof, wherein:

one of L′, L″, and L′″ in formula (PBD5′), (PBD6′), (PBD7′), (PBD8′),(PBD5), (PBD6), (PBD7) or (PBD8) is represented by the followingformula:

—Z₁—P₁—Z₂—R_(x1)—C(═O)—  (A′), or

—N(R^(e))—R_(x1)—C(═O)—  (D′);

and the other two are each independently selected from —H, an optionallysubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl havingfrom 1 to 10 carbon atoms, a polyethylene glycol unit—(CH₂CH₂O)_(n)—R^(c), halogen, guanidinium [—NH(C═NH)NH₂], —OR, —NR′R″,—NO₂, —NR′COR″, —SR, —SOR′, —SO₂R′, —SO₃H, —OSO₃H, —SO₂NR′R″, cyano, anazido, —COR′, —OCOR′, and —OCONR′R″;

one of Q₂ and Q₂′ in formula (PBD9′), (PBD10′), (PBD9) or (PBD10) isrepresented by the following formula:

—Z₁—P₁—Z₂—R_(x1)—C(═O)—  (A′),

—N(R^(c))—R_(x1)—C(═O)—  (D′); or

—Z₁—P₁—Z₂—  (E′),

and the other one is selected from —H, —R, —OR, —NR′R″, —NO₂,—NR′(C═O)OR″, —SR, or —NO₂;

one of the Z₁ and Z₂ is —C(═O)—, and the other is —NR₅—;

P₁ is an amino acid residue or a peptide containing between 2 to 20amino acid residues;

R_(x1) is an optionally substituted linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms;

R^(e) is —H, a linear, branched or cyclic alkyl, alkenyl or alkynylhaving 1 to 10 carbon atoms or —(CH₂—CH₂—O)_(n)—R^(k), wherein R^(k) isa —H, a linear, branched cyclic alkyl having 1 to 6 carbon atoms,optionally bearing a secondary amino (e.g., —NHR¹⁰¹) or tertiary amino(—NR¹⁰¹R¹⁰²) group or a 5- or 6-membered nitrogen containingheterocycle, wherein R¹⁰¹ and R¹⁰² are each independently a linear,branched, or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbonatoms;

R^(c) is —H or an optionally substituted linear or branched alkyl having1 to 4 carbon atoms;

R₅ for each occurrence is independently —H or an optionally substitutedlinear or branched alkyl having 1 to 10 carbon atoms.

R_(a)″ and R_(b)″ are the same or different, and are selected from —Hand -Me; and

n′ is selected from 0, 1, 2 and 3; and the remaining variables are asdescribed in the 25^(th) specific aspect.

In a 27^(th) specific aspect, one of L′, L″ and L′″ in formula (PBD5′),(PBD6′), (PBD7′), (PBD8′), (PBD5), (PBD6), (PBD7) or (PBD8) isrepresented by formula (A′) or (D′) and the other two are —H, an linearor branched alkyl having from 1 to 6 carbon atoms, halogen, —OH,(C₁-C₆)alkoxy or —NO₂; or one of Q₂ and Q₂′ in formula (PBD9′),(PBD10′), (PBD9) or (PBD10) is represented by formula (A′), (D′) or(E′); and the other is —H, an linear or branched alkyl having from 1 to6 carbon atoms, halogen, —OH, (C₁-C₆)alkoxy or —NO₂; and the remainingvariables are as described in the 26^(th) specific aspect.

In a 28^(th) specific aspect, L″ and L′″ are both —H; and L′ in formula(PBD5′), (PBD6′), (PBD7′), (PBD8′), (PBD5), (PBD6), (PBD7) or (PBD8) isrepresented by the following formula:

—NR₅—P₁—C(═O)—(CR_(a)R_(b))_(s)—C(═O)—  (B1′);

—NR₅—P₁—C(═O)—Cy-(CR_(a)R_(b))_(s1′)—C(═O)—  (B2′);

—C(═O)—P₁—NR₅—(CR_(a)R_(b))_(s)—C(═O)—  (C1′), or

—C(═O)—P₁—NR₅-Cy-(CR_(a)R_(b))_(s1′)—C(═O)—  (C2′);

one of Q₂ and Q₂′ in formula (PBD9′), (PBD10′), (PBD9) or (PBD10) isrepresented by the following formula:

—NR₅—P₁—C(═O)—(CR_(a)R_(b))_(s)—C(═O)—  (B1′);

—NR₅—P₁—C(═O)—Cy-(CR_(a)R_(b))_(s1′)—C(═O)—  (B2′);

—C(═O)—P₁—NR₅—(CR_(a)R_(b))_(s)—C(═O)—  (C1′),

—C(═O)—P₁—NR₅-Cy-(CR_(a)R_(b))_(s1′)—C(═O)—  (C2′);

—NR₅—P₁—C(═O)—  (E1′); or

—C(═O)—P₁—NR₅—  (E2′);

wherein:

R_(a) and R_(b), for each occurrence, are each independently —H,(C₁-C₃)alkyl or a charged substituent or an ionizable group Q;

s is an integer from 1 to 6;

s1′ is 0 or an integer from 1 to 6; and

Cy is a cyclic alkyl having 5 or 6 ring carbon atoms optionallysubstituted with halogen, —OH, (C₁-C₃)alkyl, (C₁-C₃)alkoxy, orhalo(C₁-C₃)alkyl; and the remaining variables are as described in the27^(th) specific aspect. In some embodiments, R_(a) and R_(b) are bothH; Cy in formulas (B2′) and (C₂′) is cyclohexane; R₅ is H or Me; and s1′is 0 or 1.

In a 29^(th) specific aspect, P₁ is a peptide containing 2 to 10 aminoacid residues; and the remaining variables are as described in the28^(th) specific aspect. In some embodiments, P₁ is a peptide containing2 to 5 amino acid residues. In some embodiments, P₁ is Gly-Gly-Gly,Ala-Val, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit,Ile-Cit, Trp, Cit, Phe-Ala, Phe-N⁹-tosyl-Arg, Phe-N⁹-nitro-Arg,Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu,Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ IDNO: 2), Gly-Phe-Leu-Gly (SEQ ID NO: 3), Val-Arg, Arg-Arg, Val-D-Cit,Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit,D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala,D-Ala-D-Ala, Ala-Met, Gln-Val, Asn-Ala, Gln-Phe and Gln-Ala. In someembodiments, P₁ is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala,or D-Ala-D-Ala.

In a 30^(th) specific aspect, for the methods described in any one ofthe 25^(th), 26^(th), 27^(th), 28^(th) or 29^(th) specific aspect andembodiments described therein, R₆ is —OMe; X₁′ and Y₁′ are both —H; andA and A′ are —O—.

In a 31^(st) specific aspect, for the imine-containing cytotoxic agentof formula (A) or the conjugate of formula (B), D is represented by thefollowing structural formula:

or a pharmaceutically acceptable salt thereof.

In 32^(nd) specific aspect, for the imine-containing cytotoxic agent offormula (A) or the conjugate of formula (B), -L- is represented by thefollowing structural formula:

or a pharmaceutically acceptable salt thereof, wherein:

s1 is the site covalently linked to D; s2 is the site covalently linkedto the maleimide group;

R₂₃ and R₂₄, for each occurrence, are independently H or an optionallysubstituted alkyl;

m′ is an integer between 0 and 10; and

R^(h′) is H or an optionally substituted alkyl; and the remainingvariables are as describe above in the 25^(th), 26^(th), 27^(th),28^(th), 29^(th), 30^(th), 31^(st) or 32^(nd) specific aspect.

In some embodiments, R₂₃ and R₂₄ are both H; and m′ is an integerbetween 1 and 6.

In some embodiments, R^(h)′ is H.

In some embodiments, L is represented by the following structuralformula:

In a 34^(th) specific aspect, the imine-containing cytotoxic agent isrepresented by the following formula:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the imine-containing cytotoxic agent is representedby the following formula:

pharmaceutically acceptable salt thereof; the modified imine-containingcytotoxic agent is represented by the following formula:

pharmaceutically acceptable salt thereof; and the conjugate isrepresented by the following formula:

or a pharmaceutically acceptable salt thereof.

In a 35^(th) specific aspect, for the imine-containing cytotoxic agentof formula (A) or the conjugate of formula (B), D is represented by thefollowing formula:

or a pharmaceutically acceptable salt thereof, wherein:

X₁′ is selected from the group consisting of —H, —OH, a substituted orunsubstituted linear, branched or cyclic alkyl, alkenyl or alkynylhaving from 1 to 10 carbon atoms, phenyl, and an amine-protecting group;

Y₁′ is selected from the group consisting of —H, an oxo group, asubstituted or unsubstituted linear, branched or cyclic alkyl, alkenylor alkynyl having from 1 to 10 carbon atoms;

one of Q₂ and Q₂′ in formula (PBD15) or (PBD16) is —W′—R^(x)—S—; and theother is selected from —H, an optionally substituted linear, branched orcyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, apolyethylene glycol unit —(CH₂CH₂O)_(n)—R^(c), halogen, guanidinium[—NH(C═NH)NH₂], —OR, —NR′R″, —NO₂, —NR′COR″, —SR, —SOR′, —SO₂R′, —SO₃H,—OSO₃H, —S₂NR′R″, cyano, an azido, —COR′, —OCOR′, and —OCONR′R″;

W′ is absent, or selected from —O—, —N(R^(e))—, —N(R^(e))—C(═O)—,—N(C(═O)R^(e))—, —S— or —CH₂—S—, —CH₂NR^(e)—;

R^(x) is absent or selected from a linear, branched or cyclic alkylhaving 1 to 10 carbon atoms;

R^(e) is —H, a linear, branched or cyclic alkyl, alkenyl or alkynylhaving 1 to 10 carbon atoms or —(CH₂—CH₂—O)_(n)—R^(k), wherein R^(k) isa —H, a linear, branched cyclic alkyl having 1 to 6 carbon atoms,optionally bearing a secondary amino (e.g., —NHR¹⁰¹) or tertiary amino(—NR¹⁰¹R¹⁰²) group or a 5- or 6-membered nitrogen containingheterocycle, wherein R¹⁰¹ and R¹⁰² are each independently a linear,branched, or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbonatoms;

n is an integer from 1 to 24;

R₆ is —H, —R, —OR, —SR, —NR′R″, —NO₂, or halogen; and

R is —H, an optionally substituted linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms or a PEG group—(CH₂CH₂O)_(n)—R^(c), wherein n is an integer from 1 to 24, and R^(c) isa linear or branched alkyl having 1 to 4 carbon atoms;

R′ and R″ are each independently selected from —H, —OH, —OR, —NRR^(g′),—COR, an optionally substituted linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms, an optionallysubstituted aryl having from 6 to 18 carbon atoms, an optionallysubstituted 3- to 18-membered heterocyclic ring having 1 to 6heteroatoms selected from O, S, N and P, a PEG group—(CH₂CH₂O)_(n)—R^(c), and R^(g′) is —H, an optionally substitutedlinear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10carbon atoms or a PEG group —(CH₂CH₂O)_(n)—R^(c).

In a36^(th) specific aspect, for formula (PBD11′), (PBD12′), (PBD13′),(PBD14′), (PBD15′), (PBD16′), (PBD11), (PBD12), (PBD13), (PBD14),(PBD15) or (PBD16), X₁′ and Y₁′ are both —H;

R₆ is —OMe;

W′ is —N(R^(e))— or —N(R′)—C(═O)—;

R^(e) is —H, a linear or branched alkyl having 1 to 4 carbon atoms or—(H₂—CH₂—O)_(n)—R^(k), wherein R^(k) is a —H, a linear or branched alkylhaving 1 to 4 carbon atoms;

n is an integer from 2 to 6; and

R^(x) is a linear or branched alkyl having 1 to 6 carbon atoms; and theremaining variables are as described in the 35^(th) specific aspect.

In a 37^(th) specific aspect, for the imine-containing cytotoxic agentof formula (A) or the conjugate of formula (B), L is represented by thefollowing structural formula:

or a pharmaceutically acceptable salt thereof, wherein:

s1 is the site covalently linked to D, and s2 is the site covalentlylinked to the maleimide group;

E is —(CR₁₀R₁₁)_(q)—, cycloalkyl, or cycloalkylalkyl;

Z is absent, —SO₂NR₉—, —NR₉SO₂—, —C(═O)—NR₉—, —NR₉—C(═O)—, —C(═O)—O—,—O—C(═O)—, —C(═O)—NR₉—(CH₂CH₂O)_(p)—, —NR₉—C(═O)—(CH₂CH₂O)_(p)—,—(OCH₂CH₂)_(p)—C(═O)NR₉—, or —(OCH₂CH₂)_(p)—NR₉—C(═O)—;

p is an integer from 1 to 24;

Q is H, a charged substituent, or an ionizable group;

R₉, R₁₀, R₁₁, R₁₂, and R₁₃, for each occurrence, are independently H oran optionally substituted alkyl; and, q and r, for each occurrence, areindependently an integer between 0 and 10; and the remaining variablesare as described in the 35^(th) or 36^(th) specific aspect.

In some embodiments, E is —(CR₁₀R₁₁)_(q)—. In some embodiments, Z is—C(═O)—NR₉— or —NR₉—C(═O)—. In some embodiments, R₉ is —H. In someembodiments, R₉, R₁₀, R₁₁, R₁₂, and R₁₃ are all H; and q and r are eachindependently an integer between 1 and 6.

In some embodiments, for -L- described in the 37 specific aspect:

R₁₂ and R₁₃, for each occurrence, are each independently H or(C₁-C₃)alkyl;

Q is H or —S₃H or a pharmaceutically acceptable salt thereof;

Z is —C(═O)—NR₉— or —NR₉—C(═O)—;

R₉ is H or (C₁-C₃)alkyl;

E is —(CR₁₀R₁₁)_(q)—.

R₁₀ and R₁₁, for each occurrence, are independently H or (C₁-C₃)alkyl;and

q and r are each an integer from 1 to 5.

In a 38^(th) specific aspect, for the imine-containing cytotoxic agentof formula (A) or the conjugate of formula (B), L is represented by anyone of the following structural formulae:

or a pharmaceutically acceptable salt thereof, and the remainingvariables are as described in the 35^(th) or 36^(th) specific aspect.

In a 39^(th) specific aspect, for the imine-containing cytotoxic agentof formula (A) or the conjugate of formula (B), L is represented by thefollowing structural formula:

or a pharmaceutically acceptable salt thereof, wherein:

s1 is the site covalently linked to D; s2 is the site covalently linkedto the maleimide group;

R₁₉, R₂₀, R₂₁ and R₂₂, for each occurrence, are independently H or anoptionally substituted alkyl;

m and n are each independently an integer between 0 and 10;

R^(h) is H or an optionally substituted alkyl;

P_(L) is an optionally substituted alkylene, —(CH₂—CH₂—O)_(j)— (whereinthe oxygen atom is connected to the —(C═O)— group connected to P), anamino acid residue or a peptide containing 2 to 20 amino acid residues;and

j is an integer from 1 to 24; and the remaining variables are asdescribed in the 35^(th) or 36^(th) specific aspect.

In some embodiments, R₁₉, R₂₀, R₂₁ and R₂₂ are each H; and m and n areeach independently an integer between 1 and 6.

In some embodiments, P_(L) is a peptide containing 2 to 10 amino acidresidues. More specifically, P_(L) is a peptide containing 2 to 5 aminoacid residues. Even more specifically, P_(L) is selected from the groupconsisting of: Ala-Val, Val-Ala, Val-Cit, Cit-Val. Val-Lys, Phe-Lys,Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala,Phe-N⁹-tosyl-Arg, Phe-N⁹-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys,Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2), Gly-Phe-Leu-Gly (SEQ ID NO:3), Val-Arg, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit,D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg,D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met,Met-Ala, Gln-Val, Asn-Ala, Gln-Phe, Gln-Ala, Gly-Gly-Gly, Ala-Ala-Ala,D-Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-D-Ala, Ala-Val-Cit, Ala-Val-Ala,and β-Ala-Gly-Gly-Gly.

In some embodiments, L is represented by the following structuralformula:

or a pharmaceutically acceptable salt thereof.

In a 40^(th) specific embodiment, for methods described herein, theimine-containing cytotoxic agent is represented by the followingformula:

or a pharmaceutically acceptable salt thereof, wherein:

the dotted lines indicate the optional presence of a double bond;

R^(3′) is a C₃₋₁₂ alkylene group,

each X′, for each occurrence, is independently —O—, —S— or —N(H)—,

each R² is independently selected from —H, —OH, —CN, —R^(1′), —OR^(1′),—O—SO₂—R^(1′), —CO₂R^(1′), —COR^(1′), or halo, or both R² takentogether, are ═O, ═CH₂, ═CH—R^(a), or ═C(R_(a))₂;

each R^(2′) is independently selected from —H, —OH, —CN, —R^(1′),—OR^(1′), —O—SO₂—R^(1′), —CO₂R^(1′), —COR^(1′) or halo;

R^(4b) is a leaving group selected from —OR^(6′), —OCOR^(4′),—OCOOR^(4′), —OCONR^(4′)R^(5′), —NR^(4′)R^(5′), —NR^(4′)COR^(5′),—NR^(4′)NR^(4′)R^(5′), an optionally substituted 5- or 6-memberednitrogen-containing heterocycle (e.g., piperidine, tetrahydropyrrole,pyrazole, morpholine), a guanidinum represented by—NR^(4′)(C═NH)NR^(4′)R^(5′), an amino acid, or a peptide represented by—NR^(6′)COP′, wherein P′ is an amino acid or a polypeptide containingbetween 2 to 20 amino acid units, —SR^(6′), —SOR^(4′), —SO₂M, —SO₃M,—OSO₃M, halogen, cyano and an azido;

R^(L) is linker bearing a maleimide moiety that can form a covalent bondwith a cell binding agent (CBA);

R⁶ and R⁹ are independently selected from —H, —R^(1′), —OH, —OR^(1′),—SH, —SR^(1′), —NH₂, —NHR^(1′), —NR^(1′)R^(3′), —NO₂, Me₃Sn and halo;and,

R^(1′) and R^(3′) are each independently selected from optionallysubstituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl or C₅₋₂₀ aryl groups, andoptionally in relation to the group —NR^(1′)R^(3′), R^(1′) and R^(3′)together with the nitrogen atom to which they are attached form anoptionally substituted 4-, 5-, 6- or 7-membered heterocyclic ring; orany pair of adjacent groups from R⁶ to R⁹ together form a group—O—(CH₂)_(p)—O—, where p is 1 or 2;

R^(4′) and R^(5′) are each independently selected from —H, —OH,—OR^(6′), —NHR^(6′), —NR^(6′) ₂, —COR^(6′), an optionally substitutedlinear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10carbon atoms, a polyethylene glycol unit —(CH₂CH₂O)_(n)—R^(b), or anoptionally substituted 3- to 18-membered heterocyclic ring having 1 to 6heteroatoms independently selected from O, S, N or P;

R^(6′), for each occurrence, is independently selected from the groupconsisting of —H, an optionally substituted linear, branched or cyclicalkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, apolyethylene glycol unit —(CH₂CH₂O)_(n)—R^(b), an optionally substitutedaryl having 6 to 18 carbon atoms, an optionally substituted 5- to18-membered heteroaryl ring containing one or more heteroatomsindependently selected from nitrogen, oxygen, or sulfur, and anoptionally substituted 3- to 18-membered heterocyclic ring containing 1to 6 heteroatoms independently selected from O, S, N or P;

R^(a) is independently selected from —R^(1′), —CO₂R^(1′), —COR^(1′),—CHO, —CO₂H, or halo;

R^(b) is —H or a substituted or unsubstituted linear or branched alkylhaving 1 to 4 carbon atoms;

M is H or a pharmaceutically acceptable cation; and

n is an integer from 1 to 24.

In a 41^(st) specific aspect, for the methods described herein, theimine-containing cytotoxic agent is represented by the followingformula:

or a pharmaceutically acceptable salt thereof, wherein R^(3″) is aC₃₋₅alkylene; and the remaining variables are as described above in the40^(th) specific aspect.

In a 42^(nd) specific aspect, for formula (PBD17′), (PBD18′), (PBD19′),(PBD20′), (PBD17), (PBD18), (PBD19) or (PBD20), R^(L) is

wherein L¹ is a cleavable linker, A is a connecting group bearing amaleimide capable of connecting L¹ to the cell binding agent, L² is acovalent bond or together with —OC(═O)— forms a self-immolative linker;and the remaining variables are as described above in the 40^(th) or41^(st) specific aspect.

In a 43^(rd) specific aspect, for R^(L) described in the 42^(nd)specific aspect, L¹ is a peptide containing 2 to 10 amino acid residues;and the remaining variables are as described above in the 42^(nd)specific aspect. More specifically, L¹ is a peptide containing 2 to 5amino acid residues. Even more specifically, L¹ is selected from thegroup consisting of Phe-Lys, Val-Ala, Val-Lys, Ala-Lys, Val-Cit,Phe-Cit, Leu-Cit, Ile-Cit, Phe-Arg, Trp-Cit, Lys-Lys, Phe-Ala,Phe-N9-tosyl-Arg, Phe-N9-nitro-Arg, Val-Arg, Arg-Val, Arg-Arg,Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg,D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Gly-Gly-Gly,Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu,Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO:1), β-Ala-Leu-Ala-Leu (SEQ IDNO:2) and Gly-Phe-Leu-Gly (SEQ ID NO:3).

In a 44^(th) specific aspect, for R^(L) described in the 42^(nd)specific aspect, —C(═O)O— and L² together form the group:

where the asterisk indicates the point of attachment to the N10position, the wavy line indicates the point of attachment to the linkerL¹, Y′ is —NH—, —O—, —C(═O)NH— or —C(═O)O—, and n is 0 to 3; and theremaining variables are as described in the 42^(nd) or 43^(rd) specificaspect.

In a 45^(th) specific aspect, for R^(L) described in the 42^(nd)specific aspect, —C(═O)O— and L² together form the group:

and the remaining variables are as described in the 42^(nd) or 43^(rd)specific aspect.

In a 46^(th) specific aspect, for R^(L) described in the 42^(nd)specific aspect, L¹ and L² together with —OC(═O)— comprise a groupselected from:

where the asterisk indicates the point of attachment to the N10position, and the wavy line indicates the point of attachment to theremaining portion of the linker L¹ or the point of attachment to A.

In a 47^(th) specific aspect, for R^(L) described in the 42^(nd)specific aspect, A is represented by one of the following:

where the asterisk indicates the point of attachment to L¹, and p is 1to 6; or

where the asterisk indicates the point of attachment to L¹, r is 0 or 1,and s is 0 to 30;

-   -   and the remaining variables are as described in the 42^(nd),        43^(rd), 44^(th), 45^(th) or 46^(th) specific aspect.

In some embodiments, p is 4 to 6; r is 1; and s is 1 to 10.

In a 48^(th) specific aspect, for methods described in the presentinvention, the imine-containing cytotoxic agent is represented by thefollowing formula:

or a pharmaceutically acceptable salt thereof, the modifiedimine-containing cytotoxic agent is represented by the followingformula:

or a pharmaceutically acceptable salt thereof, and the conjugate isrepresented by the following formula:

wherein s is 2 to 8; w is 1 or 2; and CBA is a cell-binding agentdescribed herein. In some embodiments, s is 7.

In some embodiments, for methods described in the present invention, theimine-containing cytotoxic agent is represented by the followingformula:

or a pharmaceutically acceptable salt thereof, the modifiedimine-containing cytotoxic agent is represented by the followingformula:

or a pharmaceutically acceptable salt thereof, and the conjugate isrepresented by the following formula:

wherein s is 2 to 8; w is 1 or 2; and CBA is a cell-binding agentdescribed herein. In some embodiments, s is 7.

In a 49^(th) specific aspect, for methods described in the presentinvention, the imine-containing cytotoxic agent is represented by thefollowing formula:

or a pharmaceutically acceptable salt thereof, the modifiedimine-containing cytotoxic agent is represented by the followingformula:

or a pharmaceutically acceptable salt thereof, and the conjugate isrepresented by the following formula.

or a pharmaceutically acceptable salt thereof, wherein s is 2 to 8; w is1 or 2; and CBA is a cell-binding agent described herein. In someembodiments, s is 7.

In some embodiments, for methods described in the present invention, theimine-containing cytotoxic agent is represented by the followingformula:

or a pharmaceutically acceptable salt thereof, the modifiedimine-containing cytotoxic agent is represented by the followingformula:

or a pharmaceutically acceptable salt thereof, and the conjugate isrepresented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein s is 2 to 8; w is1 or 2; and CBA is a cell-binding agent described herein. In someembodiments, s is 7.

In some embodiments, the charged substituent or an ionizable group Qdescribed in any embodiments above is i) —SO₃H, —Z′—SO₃H, —OPO₃H₂,—Z′—OPO₃H₂, —PO₃H₂, —Z′—PO₃H₂, —CO₂H, —Z′—CO₂H, —NR₁₁R₁₂, or—Z′—NR₁₁R₁₂, or a pharmaceutically acceptable salt thereof; or, ii)—N⁺R₁₄R₁₅R₁₆X⁻ or —Z′—N⁺R₁₄R₁₅R₁₆X⁻; Z′ is an optionally substitutedalkylene, an optionally substituted cycloalkylene or an optionallysubstituted phenylene; R₁₄ to R₁₆ are each independently an optionallysubstituted alkyl; and X⁻ is a pharmaceutically acceptable anion. Morespecifically, Q is —SO₃H or a pharmaceutically acceptable salt thereof.Even more specifically, Q is —SO₃Na.

Cell-Binding Agents

In some embodiments, the cell-binding agent can be used in the presentmethods is an antibody having an engineered cysteine residue (e.g., atthe EU/OU numbering position 442 of the heavy chain(s)). The engineeredCys residue can be located on one or both heavy chains of the antibody,or on one or both light chains of the antibody, or antigen-bindingportion thereof, or a combination thereof. In some embodiments, the Cysresidue is located at the EU/OU numbering position 442 of the antibodyheavy chain(s). In some embodiments, the antibody is a cysteineengineered antibody as described herein.

In some embodiments, the antibody of the present invention is amonoclonal antibody, a chimeric antibody, a humanized antibody, aresurfaced antibody, or a human antibody.

In other embodiments, the cell-binding agent is an antibody, a singlechain antibody, an antibody fragment that specifically binds to thetarget cell, a monoclonal antibody, a single chain monoclonal antibody,a monoclonal antibody fragment (or “antigen-binding portion”) thatspecifically binds to a target cell, a chimeric antibody, a chimericantibody fragment (or “antigen-binding portion”) that specifically bindsto the target cell, a domain antibody (e.g., sdAb), or a domain antibodyfragment that specifically binds to the target cell.

In yet other embodiments, the cell-binding agent is a humanizedantibody, a humanized single chain antibody, or a humanized antibodyfragment (or “antigen-binding portion”). In a specific embodiment, thehumanized antibody is huMy9-6 or another related antibody, which isdescribed in U.S. Pat. Nos. 7,342,110 and 7,557,189. In another specificembodiment, the humanized antibody is an anti-folate receptor antibodydescribed in U.S. Pat. No. 8,557,966. In yet other embodiments, thehumanized antibody is an anti-CD123 antibody described in U.S.application Ser. No. 15/195,401, filed on Jun. 28, 2016, entitled“ANTI-CD123 ANTIBODIES AND CONJUGATES AND DERIVATIVES THEREOF.” Theteachings of all these applications are incorporated herein by referencein its entirety.

In some embodiments, the cell-binding agent is a resurfaced antibody, aresurfaced single chain antibody, a resurfaced antibody fragment (or“antigen-binding portion”), or a bispecific antibody.

Yet another aspect of the invention provides a recombinant antibodycomprising a mature processed sequence of the heavy chain, light chain,or antigen-binding portion thereof, derived from any one of the subjectrecombinant antibody heavy chain (HC), light chain (LC), orantigen-binding portion thereof described herein.

For example, the recombinant antibody may be or may comprise an scFv-Fc,Fcab, mAb2, small modular immunopharmaceutical (SMIP), Genmab/unibody orduobody, minibody, IgGΔCH2, DVD-Ig, probody, intrabody, or amultispecificity antibody.

A DUOBODY® is a bispecific modified IgG1 antibody heterodimer. IgG1hinge region that generally includes (i) a stable hinge region thatcontains a CPPC sequence and is non-permissive for Fab arm exchange invivo and (ii) an IgG4-like CH3 domain that is modified to contain F405Land K409R residues, which renders it permissive for Fab arm exchange invivo. (See, for example, WO2008119353 and WO2011131746).

In some embodiments, the recombinant antibody may comprise 1, 2, 3, or 4of the mature processed sequence of the heavy chain, light chain, orantigen-binding portion thereof, each derived from any one of thesubject recombinant antibody heavy chain (HC), light chain (LC), orantigen-binding portion thereof described herein.

In other embodiments, the recombinant antibody may be a heterodimericantibody comprising a first heavy chain polypeptide and a second heavychain polypeptide, wherein the Fc region of the first heavy chainpolypeptide and the Fc region of the second heavy chain polypeptide meetat an interface, and the interface of the Fc region of the second heavychain polypeptide comprises a protuberance which is positionable in acavity in the interface of the Fc region of the first heavy chainpolypeptide. In certain embodiments, the knob-into-hole technology topromote specific pairing of heavy chains in the bi-specific antibody maybe further improved based on, for example, the CrossMab technology ofGenentech/Roche, e.g., by swapping CH1 and Kappa constant regions tofurther reduce or eliminate light chain mis-pairing.

Alternatively, similar results can also be achieved using LCheterodimers, such as Zymeworks AZYMETRIC™ heterodimeric IgG1 lightchain platform technology that fully complements multiple otherbiologics approaches, including common light chain, domain antibody, andsingle chain formats, in the development of fully bi-specificantibodies.

In some embodiments, the Fc region of the second heavy chain polypeptidehas been altered from a template/original polypeptide to encode theprotuberance, or the Fc region of the first heavy chain polypeptide hasbeen altered from a template/original polypeptide to encode the cavity,or both.

In other embodiments, the protuberance and the cavity each comprises anaturally occurring amino acid residue.

In other embodiments, the Fc region of the second heavy chainpolypeptide comprising the protuberance is generated by replacing anoriginal residue from the interface of a template/original polypeptidewith an import residue having a larger side chain volume than theoriginal residue.

In yet other embodiments, the Fc region of the second heavy chainpolypeptide comprising the protuberance is generated by a methodcomprising a step wherein nucleic acids encoding an original residuefrom the interface of said polypeptide is replaced with nucleic acidsencoding an import residue having a larger side chain volume than theoriginal.

In some embodiments, the antibody includes bispecific, multispecific,and monospecific antibody variants that include the antigen bind regionsand the heavy chain constant domain, wherein the heavy chain constantdomain is modified to include a Cys at position 442 of the EU/OUnumbering.

In other embodiments, the antibody may bind to a ligand on the targetcell, such as a cell-surface ligand, including cell-surface receptors.

Specific exemplary antigens or ligands may include renin; a growthhormone (e.g., human growth hormone and bovine growth hormone); a growthhormone releasing factor; a parathyroid hormone; a thyroid stimulatinghormone; a lipoprotein; alpha-1-antitrypsin; insulin A-chain; insulinB-chain; proinsulin; a follicle stimulating hormone; calcitonin; aluteinizing hormone; glucagon; a clotting factor (e.g., factor vmc,factor IX, tissue factor, and von Willebrands factor); an anti-clottingfactor (e.g., Protein C); an atrial natriuretic factor; a lungsurfactant; a plasminogen activator (e.g., a urokinase, a human urine ortissue-type plasminogen activator); bombesin; a thrombin; hemopoieticgrowth factor; tumor necrosis factor-alpha and -beta; an enkephalinase;RANTES (i.e., the regulated on activation normally T-cell expressed andsecreted); human macrophage inflammatory protein-1-alpha; a serumalbumin (human serum albumin); Muellerian-inhibiting substance; relaxinA-chain; relaxin B-chain; prorelaxin; a mouse gonadotropin-associatedpeptide; a microbial protein (beta-lactamase); DNase; IgE; a cytotoxicT-lymphocyte associated antigen (e.g., CTLA-4); inhibin; activin; avascular endothelial growth factor; a receptor for hormones or growthfactors; protein A or D; a rheumatoid factor; a neurotrophicfactor(e.g., bone-derived neurotrophic factor, neurotrophin-3, -4, -5,or -6), a nerve growth factor (e.g., NGF-β); a platelet-derived growthfactor; a fibroblast growth factor (e.g., aFGF and bFGF); fibroblastgrowth factor receptor 2; an epidermal growth factor; a transforminggrowth factor (e.g., TGF-alpha, TGF-β1, TGF-β2, TGF-β3, TGF-β4, andTGF-β5); insulin-like growth factor-I and -II; des(1-3)-IGF-I (brainIGF-I); an insulin-like growth factor binding protein;melanotransferrin; EpCAM; GD3; FLT3; PSMA; PSCA; MUC1; MUC16; STEAP;CEA; TENB2; an EphA receptor; an EphB receptor; a folate receptor;FOLR1; mesothelin; cripto; an alpha_(v)beta₆; integrins; VEGF; VEGFR;EGFR; transferrin receptor; IRTA1; IRTA2; IRTA3; IRTA4; IRTA5; CDproteins (e.g., CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD19, CD20,CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44,CD52, CD55, CD56, CD59, CD70, CD79, CD80. CD81, CD103, CD105, CD123,CD134, CD137, CD138, and CD152), one or more tumor-associated antigensor cell-surface receptors (see US Publication No. 2008/0171040 or USPublication No. 2008/0305044, incorporated in their entirety byreference); erythropoietin; an osteoinductive factor; an immunotoxin; abone morphogenetic protein; an interferon (e.g., interferon-alpha,-beta, and -gamma); a colony stimulating factor (e.g., M-CSF, GM-CSF,and G-CSF); interleukins (e.g., IL-1 to IL-10); a superoxide dismutase;a T-cell receptor; a surface membrane protein; a decay acceleratingfactor; a viral antigen (e.g., a portion of the HIV envelope); atransport protein, a homing receptor; an addressin; a regulatoryprotein; an integrin (e.g., CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4,and VCAM;) a tumor associated antigen (e.g., HER2, HER3 and HER4receptor); endoglin; c-Met; c-kit; 1GF1R; PSGR; NGEP; PSMA; PSCA;TMEFF2; LGR5; B7H4; and fragments of any of the above-listedpolypeptides.

In some embodiments, the cell-binding agent is an anti-folate receptorantibody. More specifically, the anti-folate receptor antibody is ahumanized antibody that specifically binds a human folate receptor 1,wherein the antibody comprises: (a) a heavy chain CDR1 comprising GYFMN(SEQ ID NO: 4); a heavy chain CDR2 comprisingRIHIPYDGDTFYNQXaa₁FXaa₂Xaa₃ (SEQ ID NO: 5); and a heavy chain CDR3comprising YDGSRAMDY (SEQ ID NO: 6); and (b) a light chain CDR1comprising KASQSVSFAGTSLMH (SEQ ID NO: 7); a light chain CDR2 comprisingRASNLEA (SEQ ID NO: 8); and a light chain CDR3 comprising QQSREYPYT (SEQID NO: 9); wherein Xaa₁ is selected from K, Q, H, and R; Xaa₂ isselected from Q, H, N, and R; and Xaa₃ is selected from G, E, T, S, A,and V. Preferably, the heavy chain CDR2 sequence comprises

(SEQ ID NO: 10) RIHPYDGDTFYNQKFQG.

In other embodiments, the anti-folate receptor antibody is a humanizedantibody or antigen binding fragment thereof that specifically binds thehuman folate receptor 1 comprising the heavy chain having the amino acidsequence of

(SEQ ID NO: 11) QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LCLSPG.

In other embodiments, the anti-folate receptor antibody is a humanizedantibody or antigen binding fragment thereof that specifically binds thehuman folate receptor 1 comprising the light chain having the amino acidsequence of

(SEQ ID NO: 12) DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; or (SEQ ID NO: 13)DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

In other embodiments the anti-folate receptor antibody is a humanizedantibody or antigen binding fragment thereof that specifically binds thehuman folate receptor 1 comprising the heavy chain having the amino acidsequence of SEQ ID NO: 11, and the light chain having the amino acidsequence of SEQ ID NO: 12 or SEQ ID NO: 13. Preferably, the antibodycomprises the heavy chain having the amino acid sequence of SEQ ID NO:11 and the light chain having the amino acid sequence of SEQ ID NO: 13(huMov19).

In other embodiments, the anti-folate receptor antibody is a humanizedantibody or antigen binding fragment thereof comprising an engineeredCys residue (e.g., C442) and a heavy chain variable domain at leastabout 90%, 95%, 99% or 100% identical to

(SEQ ID NO: 14) QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTR YDGSRAMDYWGQGTTVTVSS,and a light chain variable domain at least about 90%, 95%, 99% or 100%identical to

(SEQ ID NO: 15) DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLNISPVEAEDAATYYCQQSREY PYTFGGGTKLEIKR; or(SEQ ID NO: 16) DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREY PYTFGGGTKLEIKR.

In a specific embodiment, the humanized antibody is huMy9-6 or anotherrelated antibody, which is described in U.S. Pat. Nos. 7,342,110 and7,557,189. In another specific embodiment, the humanized antibody is ananti-folate receptor antibody (e.g., huMov19) described in U.S. Pat. No.8,577,966. In certain embodiments the humanized antibody is an anti-CD37antibody (e.g., anti-CD37-3) described in U.S. Pat. No. 8,765,917. Incertain embodiments, the humanized antibody is an anti-EGFR antibodydescribed in U.S. Pat. No. 8,790,649. In other embodiments, the antibodyis an anti-EGFR antibody. In some embodiments, the anti-EGFR antibody isa non-antagonist antibody, including, for example, the antibodiesdescribed in WO2012058592, herein incorporated by reference. In otherembodiments, the anti-EGFR antibody is a non-functional antibody, forexample, humanized ML66. More specifically, the anti-EGFR antibody ishuML66.

In some embodiments, the antibody is an anti-CD123 antibody, such as ahumanized huCD123 antibody as described in U.S. application Ser. No.15/195,401, filed on Jun. 28, 2016, entitled “ANTI-CD123 ANTIBODIES ANDCONJUGATES AND DERIVATIVES THEREOF” (entire contents, including allsequences and drawings, incorporated herein).

In a specific embodiment, the anti-CD123 antibody the antibody orantigen-binding fragment thereof comprises: a) at least one heavy chainvariable region or fragment thereof comprising three sequentialcomplementarity-determining regions (CDR) CDR1, CDR2, and CDR3,respectively, wherein, CDR1 has the sequence of SSIMH (SEQ ID NO:17),CDR2 has the sequence of YIKPYNDGTKYNEKFKG (SEQ ID NO:18), and, CDR3 hasthe sequence of EGGNDYYDTMDY (SEQ ID NO:19); and b) at least one lightchain variable region or fragment thereof comprising three sequentialcomplementarity-determining regions (CDR) CDR1, CDR2, and CDR3,respectively, wherein, CDR1 has the sequence of RASQDINSYLS (SEQ IDNO:20), CDR2 has the sequence of RVNRLVD (SEQ ID NO:21), and, CDR3 hasthe sequence of LQYDAFPYT (SEQ ID NO:22).

In another specific embodiments, the anti-CD123 antibody orantigen-binding fragment thereof comprises an engineered Cys residue(e.g., C442); an immunoglobulin heavy chain variable domain at leastabout 90%, 95%, 99% or 100% identical to

(SEQ ID NO: 23) QXQLVQSGAEVKKPGASVKVSCKASGYIFTSSIMHWVRQAPGQGLEWIGYIKPYNDGTKYNEKFKGRATLTSDRSTSTAYMELSSLRSEDTAVYYCAREGGNDYYDTMDYWGQGTLVTVSS;and an immunoglobulin light chain variable region having the amino acidsequence at least about 90%, 95%, 99% or 100% identical to

(SEQ ID NO: 24) DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRVNRLVDGVPSRFSGSGSGNDYTLTISSLQPEDFATYYCLQYDAFPYTF GQGTKVEIKR.In certain embodiments, Xaa, the second residue from the N-terminus ofSEQ ID NO: 23, is Phe. In other embodiments, Xaa is Val.

In certain embodiments, the anti-CD123 antibody or antigen-bindingfragment thereof may comprise: an immunoglobulin heavy chain regionhaving the amino acid sequence of

(SEQ ID NO: 25) QVQLVQSGAEVKKPGASVKVSCKASGYIFTSSIMHWVRQAPGQGLEWIGYIKPYNDGTKYNEKFKGRATLTSDRSTSTAYMELSSLRSEDTAVYYCAREGGNDYYDTMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLCLSPG;and an immunoglobulin light chain region having the amino acid sequenceof

(SEQ ID NO: 26) DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRVNRLVDGVPSRFSGSGSGNDYTLTISSLQPEDFATYYCLQYDAFPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

Compositions and Methods of Use

The present invention also includes the cell-binding agent-cytotoxicagent conjugates prepared by any methods of the present invention, acomposition (e.g., a pharmaceutical composition) comprising thecell-binding agent-cytotoxic agent conjugates prepared by any methods ofthe present invention and a carrier (a pharmaceutically acceptablecarrier). The present conjugates and compositions are useful forinhibiting abnormal cell growth or treating a proliferative disorder ina mammal (e.g., human). The present invention includes a method ofinhibiting abnormal cell growth or treating a proliferative disorder ina mammal (e.g., human) comprising administering to said mammal atherapeutically effective amount of the conjugates prepared by themethods of the present invention described above or a compositionthereof, alone or in combination with a second therapeutic agent.

In some embodiments, the proliferative disorder is cancer. Cancer caninclude a hematological cancer or a solid tumor. More specifically, thecancer is leukemia (e.g., acute myeloid leukemia (AML), acutelymphoblastic leukemia (ALL) such as acute B lymphoblastic leukemia(B-ALL), chronic myelogenous leukemia (CML), chronic lymphocyticleukemia (CLL)) or lymphoma, melanoma, lung cancer (e.g., non-small celllung cancer), ovarian cancer, endometrial cancer, peritoneal cancer,pancreatic cancer, breast cancer, prostate cancer, and cervical cancer.

The present invention also provides methods of treatment comprisingadministering to a subject in need of treatment an effective amount ofany of the conjugates described above.

Similarly, the present invention provides a method for inducing celldeath in selected cell populations comprising contacting target cells ortissue containing target cells with an effective amount of a cytotoxicagent comprising any of the conjugates described above. The target cellsare cells to which the cell-binding agent can bind.

If desired, other active agents, such as other anti-tumor agents, may beadministered along with the conjugate.

Suitable pharmaceutically acceptable carriers, diluents, and excipientsare well known and can be determined by those of ordinary skill in theart as the clinical situation warrants. Examples of suitable carriers,diluents and/or excipients include: (1) Dulbecco's phosphate bufferedsaline, pH about 7.4, containing or not containing about 1 mg/mL to 25mg/mL human serum albumin, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5%(w/v) dextrose; and may also contain an antioxidant such as tryptamineand a stabilizing agent such as Tween 20.

EXAMPLES Example 1. Preparation of Imine-Containing Cytotoxic Agents

The following solvents, reagents, protecting groups, moieties and otherdesignations may be referred to by their abbreviations in parenthesis:

Me methyl Et ethyl Pr propyl i-Pr isopropyl Bu butyl t-Bu tert-butyl Phphenyl Ac acetyl AcOH/ acetic acid HOAc Ala alanine aq aqueous ACN/acetonitrile CH₃CN DI deionized DCM/ dichloromethane Boc/BOCtert-butoxycarbonyl water water CH₂Cl₂ g grams DMA N,N- DIEA or N,N-dimethylacetamide DIPEA diisopropylethylamine h hour DMF N,N- EDC1-ethyl-3-(3- dimethylformamide dimethylaminopropyl) carbodiimide minminutes EtOAc ethylacetate ESI or ES electrospray ionization mgmilligrams LC liquid HPLC high-performance chromatography liquidchromatography mL milliliters mmol millimoles LCMS liquid chromatographymass spectrometry μg micrograms mmol micromoles MS mass spectrometry μLmicroliters MeOH methanol NHS N-hydroxy succinamide sat or saturated RTor rt room temperature NMR nuclear magnetic sat'd (ambient, aboutresonance 25° C.) spectroscopy THF tetrahydro TEA triethylamine RPHPLCor reverse phase high- furan (Et₃N) RP-HPLC performance liquidchromatography

A. Synthesis ofN-(2-(2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)ethyl)-N6-((S)-1-(((S)-1-((3-((((S)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)-5-((((S)-8-methoxy-6-oxo-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)adipamide,compound D1

NHS ester 1a (8.2 mg, 7.6 μmol) (prepared according to proceduresdescribed in US 2016/0082114, incorporated herein by reference) and1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (2.2 mg, 0.011 mmol)were dissolved in anhydrous dichloromethane (305 μL) at roomtemperature. DIPEA (2.66 μL, 0.015 mmol) was added and the reaction andwas stirred for 3.5 hours. The reaction mixture was concentrated and waspurified by RPHPLC (C18 column, CH₃CN/H₂O, gradient, 35% to 55%). Thedesired product fractions were frozen and lyophilized to give maleimide,compound D1 as a solid white powder (5.3 mg, 58% yield). LCMS=5.11 min(8 min method). MS (m/z): 1100.6 (M+1)⁺.

B. Synthesis ofN-(2-(2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)ethyl)-11-(3-((((S)-8-methoxy-6-oxo-11,12,12a,3-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)-5-((((S)-8-methoxy-6-oxo-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)phenyl)-13,13-dimethyl-2,5,8-trioxa-14,15-dithia-11-azanonadecan-19-amide,compound D2

To a solution of the free thiol 2a (40 mg, 0.042 mmol) and NHS4-(2-pyridyldithio)butanate (35 mg, 80% purity, 0.085 mmol) in anhydrousdichloromethane (0.5 mL) was added anhydrous diisopropylethylamine(0.015 mL, 0.085 mmol) and was stirred at room temperature for 16 hours.The reaction mixture was quenched with saturated ammonium chloride anddiluted with dichloromethane. The obtained mixture was separated in aseparatory funnel. The organic layer was washed with brine, dried overanhydrous sodium sulfate, filtered and stripped under reduced pressure.The residue was purified by semi-preparative reverse phase HPLC (C18column, CH₃CN/H₂O). The fractions that contained pure product werecombined, frozen and lyophilized to give the desired NHS ester, 2b (29.7mg, 60% yield). LCMS=9.1 min (15 min method). MS (m/z): 1157.3 (M+1)⁺.

To a solution of the NHS ester, 2b (12.3 mg, 0.011 mmol) andN-(2-aminoethyl)maleimide hydrochloride (2.0 mg, 0.011 mmol) inanhydrous dichloromethane (0.3 mL) was added DIPEA (0.0022 mL, 0.013mmol). The mixture was stirred at room temperature for 3 hours then itwas stripped under reduced pressure. The residue was purified bysemi-preparative reverse phase HPLC (C18 column, CH₃CN/H₂O). Thefractions that contained pure product were combined, frozen andlyophilized to give the desired maleimide, compound D2 (10 mg, 80%yield). LCMS=8.3 min (15 min method). MS (m/z): 1181.8 (M+1)⁺.

C. Synthesis of1-((2-(2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)ethyl)amino)-4-((5-((3-((((S)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)-5-((((S)-8-methoxy-6-oxo-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)phenyl)amino)-2-methyl-5-oxopentan-2-yl)disulfanyl)-1-oxobutane-2-sulfonicacid, compound D3

To a suspension of the free thiol, 3a (88 mg, 0.105 mmol) and1-((2,5-dioxopyrrolidin-1-yl)oxy)-1-oxo-4-(pyridin-2-yldisulfanyl)butane-2-sulfonicacid (64.0 mg, 0.158 mmol) in anhydrous dichloromethane (2.10 mL) wasadded DIPEA (55.0 μL, 0.315 mmol) under nitrogen at room temperature.The mixture stirred for 16 hours and then1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (55.6 mg, 0.315mmol), anhydrous dichloromethane (1.0 mL) and DIPEA (0.055 mL, 0.315mmol) were added. The mixture stirred for an additional 5 hours at roomtemperature upon which the reaction was concentrated in vacuo. Theresulting residue was purified by RP-HPLC (C18, CH₃CN/H₂O). Fractionscontaining desired product were frozen and lyophilized to givemaleimide, compound D3 (20 mg, 16% yield) as a white solid. LCMS=4.92min (8 min method). MS (m/z): 1158.6 (M+1)⁺.

D. Synthesis ofN-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-11-(3-((((S)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)-5-((((S)-8-methoxy-6-oxo-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)phenyl)-2,5,8-trioxa-1-azapentadecan-15-amide,compound D4

To a solution of NHS ester, 4a (5 mg, 4.82 μmol) and1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (1.7 mg, 9.64 μmol)in anhydrous dichloromethane (200 μL) was added DIPEA (1.512 μL, 8.68μmol) under nitrogen. The mixture was stirred at room temperature for 4hours and then concentrated in vacuo. The resulting residue was purifiedby RP-HPLC (C18, CH₃CN/H₂O). Fractions containing desired product werefrozen and lyophilized to give maleimide, compound D4 (3.5 mg, 68%yield). LCMS=4.61 min (15 min method). MS (m/z): 1062.8 (M+1)⁺.

Example 2. Selective Sulfonation of Imine-Containing Cytotoxic AgentBearing Maleimide Group

To a mixture of 50 mM sodium succinate pH 3.3 (116.5 mL) and DMA (98.5mL) was added D1 (263.6 mg) dissolved in 21.4 mL of DMA. Subsequently3.4 mL of a 100 mM sodium bisulfite solution (1.4 equivalents) in watercontaining 1 v/v % DMA was introduced into the reaction. The homogenousmixture was allowed to react for 2 h at 25° C., at which timecompleteness of the reaction was assayed by UPLC-MS. The reactionmixture is suitable for conjugation without further purification. Asshown in FIG. 1, UPLC-MS analysis of the reaction mixture shows 92.5%imine-sulfo D1, 1.9% unreacted D1, 0.8% maleimide-sulfo D1, and 4.8%di-sulfo D1. ESI-MS negative ion mode [M−H]⁻ calcd. for imine-sulfo D1(C₆₀H₆₂N₉O₁₅S⁻): 1180.41; found: 1180.03.

Example 3. Effect of Sodium Bisulfite on Selective Sulfonation

As indicated in Table 1, to the required volume of 50 mM sodiumsuccinate pH 3.3 buffer was added the following reagents in thefollowing order: DMA (38.8 uL), the required volume of 20 or 40 mMaqueous sodium bisulfite stock containing 1 v/v % DMA, and 8.9 mM D1 inDMA (11.2 uL). The resulting reaction mixture containing 50% DMA byvolume was allowed to react for 20 h at 25° C. The reaction productswere analyzed by UPLC-MS. The relative abundances of the observeddi-sulfo D1, maleimide-sulfo D1, imine-sulfo D1, and unsulfonated D1products are shown in Table 1.

TABLE 1 DI Reaction products (%) Sodium Sodium Bisulfite stock TotalSuccinate Stock Volume reactive buffer Total concentration added Di-Maleimide- Imine- maleimide Reaction (uL) equivalents (mM) (uL) sulfosulfo sulfo Unsulfonated remaining 1 46 0.8 20 4 0.6 1.4 35.9 62.1 98.02 45 1.0 20 5 1.1 1.4 53.4 44.2 97.6 3 44 1.2 20 6 2.0 1.2 69.6 27.296.8 4 43 1.4 20 7 2.6 1.1 79.1 17.2 96.3 5 42 1.6 20 8 5.0 1.1 85.7 8.293.9 6 41 1.8 20 9 8.3 1.2 84.6 5.9 90.5 7 45 2.0 40 5 8.7 0.8 88.2 2.390.5 8 44 2.4 40 6 13.0 0.9 83.9 2.2 86.1 9 43 2.8 40 7 16.4 1.1 79.82.7 82.5 10 42 3.2 40 8 19.8 1.5 75.8 2.9 78.7 11 41 3.6 40 9 22.9 1.871.9 3.4 75.3

Example 4. Effect of pH on Selective Sulfonation

As detailed in Table 2, to 44.0 uL of 50 mM sodium succinate buffer withthe indicated pH was added the following reagents in the followingorder: 20 mM aqueous sodium bisulfite stock (6.0 uL) containing 1% v/vDMA, DMA (38.8 uL), and 8.9 mM D1 in DMA (11.2 uL). The resultingreaction mixture containing 5000 DMA by volume was allowed to react for4 h at 25° C. The reaction products were analyzed by UPLC-MS. Therelative abundances of the observed di-sulfo, maleimide-sulfo,imine-sulfo, and unsulfonated D1 products are shown in Table 2.

TABLE 2 DGN549-C Reaction products (%) Sodium Total Sodium Bisulfitereactive Succinate (Total Di- Maleimide- Imine- maleimide Reactionbuffer pH equivalents) sulfo sulfo sulfo Unsulfonated remaining 1 2.91.2 0.7 1.0 68.5 29.8 98.3 2 3.1 1.2 0.8 1.3 65.0 33.0 97.9 3 3.3 1.21.1 1.7 66.9 30.3 97.1 4 3.4 1.2 1.3 1.4 66.5 30.7 97.3 5 3.7 1.2 2.32.7 65.6 29.4 94.9

Example 5. Selective Sulfonation with or without Buffer

As detailed in Table 3, to 21.6 uL of DMA was added 22.0 uL of water or50 mM sodium succinate buffer with the indicated pH, 20 mM aqueoussodium bisulfite stock (3.0 uL) containing 1 v/v % DMA, and 14.5 mM D1in DMA (3.4 uL). The resulting reaction mixture containing 50% DMA byvolume was allowed to react for 6 h at 25° C. The reaction products wereanalyzed by UPLC-MS. The relative abundances of the observed di-sulfo,maleimide-sulfo, imine-sulfo, and unsulfonated D1 products are shown inTable 3.

TABLE 3 DI Reaction products (%) Sodium Total Sodium Bisulfite reactiveSuccinate (Total Di- Maleimide- Imine- maleimide Reaction buffer pHequivalents) sulfo sulfo sulfo Unsulfonated remaining 1 3.5 1.2 0.8 0.586.9 11.8 98.7 2 4.0 1.2 4.8 2.9 70.0 22.8 92.8 3 water only 1.2 2.8 1.875.8 19.6 95.4

Similarly, as detailed in Table 4, to 47.6 uL of DMA was added 55.6 uLof water or pH 4.75 50 mM sodium succinate buffer, 20 mM aqueous sodiumbisulfite stock (6.9 uL) containing 1 v/v % DMA, and 8.4 mM D1 in DMA(14.9 uL). The resulting reaction mixture containing 50% DMA by volumewas allowed to react for 24 h at 25° C. The reaction products wereanalyzed by UPLC-MS (see FIG. 2). The relative abundances of theobserved di-sulfo, maleimide-sulfo, imine-sulfo, and unsulfonatedDGN549-C products are indicated in Table 4.

TABLE 4 DI Reaction products (%) Sodium Total Sodium Bisulfite reactiveSuccinate (Total Di- Maleimide- Imine- maleimide Reaction buffer pHequivalents) sulfo sulfo sulfo Unsulfonated remaining 1 4.75 1.1 9.840.7 13.4 36.1 49.5 3 water only 1.1 1.5 0.9 73.8 23.8 97.6

In another experiment, as detailed in Table 5, to 24.6 uL of DMA wasadded the indicated volume of water, the indicated volume of 20 mMaqueous sodium bisulfite stock. The pH of these solutions was asindicated in Table 5. To these mixtures was added 11.2 mM D1 in DMA (5.4uL). The resulting reaction mixtures containing 50% DMA by volume wereallowed to react for 1 to 2 h at 25° C. The reaction products wereanalyzed by UPLC-MS. The relative abundances of the observed di-sulfo,maleimide-sulfo, imine-sulfo, and unsulfonated D1 products are indicatedin Table 5.

TABLE 5 DGN549-C Reaction products (%) Sodium Bisulfite stock TotalStock Volume Observed reactive Water Total concentration added reactionDi- Maleimide- Imine- maleimide Reaction (uL) equivalents (mM) (uL) pHsulfo sulfo sulfo Unsulfonated remaining 1 21.0 3.0 20 9.0 4.2 6.9 —90.9 2.2 93.1 2 15.0 5.0 20 15.0 4.2 20.5 — 78.0 1.5 79.5 “—” means notobserved by UPLC.

Example 6. Preparation of Antibody-Cytotoxic Agent Conjugates

The sulfonation reaction mixture (240 mL, 3.5 equiv.) prepared accordingto Example 2 was subsequently introduced into a 50 mM potassiumphosphate pH 6.0 solution containing 10 g of anti-CD123 antibody withreduced C442 engineered cysteine residues. At a final concentration of 2mg/mL antibody and 15 v/v % DMA, the conjugation reaction was allowed toproceed for 18 h at 25° C. SEC analysis of the reaction product givesADC with a DAR (drug to antibody ratio) of 1.9 and a % HMW (percentageof high molecule weight species) of 4.4% vs. 3.7% prior to conjugation.

Conjugates with two other humanized monoclonal antibodies with reducedC442 engineered cysteine residues were also prepared according tosimilar procedures described above.

Example 7. Selective Sulfonation of Imine-Containing PBD Dimers BearingMaleimide Group

To 21.2 μL of 50 mM sodium succinate pH 3.3 buffer was added in thefollowing order: 20 μL of DMA, 3.8 μL of 20 mM aqueous sodium bisulfitestock, and 5.0 μL of 10.0 mM talirine in DMA. This corresponds to 1.5equivalents of bisulfite with respect to talirine. The resultingreaction mixture containing 50% DMA by volume was allowed to react for 4h at 25° C. The reaction products were analyzed by UPLC-MS. The relativeabundances of the observed imine di-sulfo, total imine mono-sulfo,unsulfonated, and total maleimide-sulfo products were determined asindicated in Table 6. ESI-MS calcd. for talirine (C₆₀H₆₅N₈O₁₂ ⁺) [M+H]⁺1089.4716, found 1089.4716; calcd. for imine mono-sulfonated talirine(C₆₀H₆₅N₈O₁₅S⁻) [M−H]⁻ 1169.4296, found 1169.4345; calcd. for iminedi-sulfonated talirine (C₆₀H₆₇N₈O₁₈S₂ ⁻) [M−H]⁻ 1251.4020, found1251.4053. A representative chromatogram (absorbance at 330 nm) of thefinal reaction mixture is shown in FIG. 3. Structures of the identifiedreaction products are

TABLE 6 Sodium Bisulfite stock Talirine Reaction products (%) SodiumTotal Stock Total imine Total Total reactive Succinate equivalentsconcentration Volume Imine di- mono- maleimide- maleimide Reactionbuffer (uL) (vs payload) (mM) added (uL) sulfo sulfo sulfo speciesUnsulfonated remaining talirine 21.2 1.5 20 3.8 57.3 39.0 <1 3.7 >99.0

Example 8. Selective Sulfonation of Imine-Containing IGN Dimers BearingMaleimide Group

As indicated in Table 7, to the required volume of 50 mM sodiumsuccinate pH 3.3 buffer was added in the following order: DMA (21.2 uL),the required volume of 20 mM aqueous sodium bisulfite stock, and 13.2 mMD5 in DMA (3.8 uL). The resulting reaction mixture containing 50% DMA byvolume was allowed to react for 4 h at 25° C. The reaction products wereanalyzed by UPLC-MS. The relative abundances of the observed iminedi-sulfo, imine mono-sulfo, unsulfonated, and total maleimide-sulfonatedproducts were determined as indicated in Table 7. ESI-MS calcd. for D5(C₆₀H₆₀N₉O₁₂ ⁺) [M+H]⁺ 1098.4356, found 1098.4351; calcd. for iminemono-sulfonated D5 (C₆₀H₆₀N₉O₁₅S⁻) [M−H]⁻ 1178.3935, found 1178.4006;calcd. for imine di-sulfonated D5 (C₆₀H₆₂N₉O₁₈S₂ ⁻) [M−H]⁻ 1260.3660,found 1260.3704; calcd. for imine di-sulfonated maleimide sulfonated D5(C₆₀H₆₄N₉O₂₁S₃ ⁻) [M−2H]²⁻ z=2 670.6653, found 670.6693. Representativechromatograms (absorbance at 330 nm) of the final reaction mixture with2.0 and 2.5 equivalents of bisulfite are shown in FIGS. 4A and 4B.Structures of the identified reaction products are shown below.

TABLE 7 Sodium Bisulfite stock IGN148-mal Reaction products (%) SodiumTotal Stock Total imine Total Total reactive Succinate equivalentsconcentration Volume Imine di- mono- maleimide- maleimide Reactionbuffer (uL) (vs payload) (mM) added (uL) sulfo sulfo sulfo speciesUnsulfonated remaining 1 21.2 1.5 20 3.8 41.9 46.9 1.4 9.7 98.6 2 20.02.0 20 5.0 76.0 20.3 1.9 1.8 98.1 3 18.7 2.5 20 6.3 83.3 6.7 10.0 — 90.0

1-11. (canceled)
 12. A method of preparing an antibody-cytotoxic agentconjugate comprising the steps of: (a) reacting an imine-containingcytotoxic agent represented by the following formula:

or a pharmaceutically acceptable salt thereof, with sodium bisulfitesalt or sodium metabisulfite salt in a mixture of an organic solvent andwater in the absence of a buffer to form a modified cytotoxic agentcomprising a modified imine moiety represented by the following formula:

or a pharmaceutically acceptable salt thereof; and (b) reacting themodified cytotoxic agent with an antibody to form the antibody-cytotoxicagent conjugate.
 13. The method of claim 12, wherein 0.5 to 5equivalents of the bisulfite salt or 0.25 to 2.5 equivalents of themetabisulfite salt is reacted with 1 equivalent of the imine-containingcytotoxic agent.
 14. The method of claim 13, wherein 0.8 to 2.0equivalents of the bisulfite salt or 0.4 to 1.0 equivalents of themetabisulfite salt is reacted with 1 equivalent of the imine-containingcytotoxic agent.
 15. The method of claim 13, wherein 1.1 to 1.6equivalents of the bisulfite salt or 0.55 to 0.8 equivalents of themetabisulfite salt is reacted with 1 equivalent of the imine-containingcytotoxic agent.
 16. The method of claim 13, wherein 1.4 equivalents ofthe bisulfite salt or 0.7 equivalent of the metabisulfite salt isreacted with 1 equivalent of the imine-containing cytotoxic agent.17-20. (canceled)
 21. The method of claim 12, wherein the reaction ofstep (a) is carried out in a mixture of dimethylacetamide (DMA) andwater.
 22. The method of claim 12, wherein the reaction of step (a) iscarried out in a mixture of DMA and water, wherein the volume ratio ofDMA and water is 1:1. 23-25. (canceled)
 26. The method of claim 12,wherein in step (a), the imine-containing cytotoxic agent is reactedwith sodium bisulfite.
 27. The method of claim 12, wherein in step (a),the imine-containing cytotoxic agent is reacted with sodiummetabisulfite.
 28. The method of claim 12, wherein the modifiedcytotoxic agent is not purified before reacting with the cell-bindingagent in step (b).
 29. (canceled)
 30. The method of claim 12, whereinthe reaction of step (b) is carried out at a pH of 4 to
 9. 31. Themethod of claim 30, wherein the reaction of step (b) is carried out at apH of 5 to 8.5.
 32. The method of claim 30, wherein the reaction of step(b) is carried out at a pH of 5.5 to 6.5.
 33. The method of claim 12,wherein the conjugate is purified by tangential flow filtration to yielda purified conjugate.
 34. The method of claim 33, wherein the purifiedconjugate is formulated in a formulation buffer comprising a bisulfitesalt.
 35. The method of claim 34, wherein the formulation buffercomprises 10 to 200 μM of sodium bisulfite.
 36. The method of claim 33,wherein the formulation buffer comprises 40 to 80 μM of sodiumbisulfite.
 37. The method of claim 33, wherein the formulation buffercomprises 50 μM of sodium bisulfite.
 38. The method of claim 34, whereinthe formulation buffer is at a pH of 4 to
 5. 39. The method of claim 38,wherein the formulation buffer is at a pH of 4.2. 40-167. (canceled)